Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease

ABSTRACT

The present invention provides anti-human ICOS antibodies with increased effector function. The invention further relates to pharmaceutical compositions, immunotherapeutic compositions, and methods using therapeutic antibodies that bind to the human ICOS antigen and that may mediate ADCC, CDC, and/or antibody-dependent phagocytosis (opsonisation) for the treatment and prevention of T cell-mediated diseases and disorders, such as, but not limited to, chronic infection, autoimmune disease or disorder, inflammatory disease or disorder, graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder.

This application claims the benefit under 35 U.S.C. §119 (e) of U.S.Provisional Application Nos. 60/916,400, filed May 7, 2007, and61/049,131, filed Apr. 30, 2008, the disclosures of each of which areincorporated herein in their entirety for all purposes.

1. INTRODUCTION

The present invention relates to anti-ICOS antibodies with enhancedeffector function. The present invention is also directed tocompositions comprising anti-ICOS antibodies with enhanced effectorfunction that may mediate one or more of the following:complement-dependent cell-mediated cytotoxicity (CDC), antigen-dependentcell-mediated-cytotoxicity (ADCC), and antibody-dependent phagocytosis(opsonisation). The present invention is further directed tocompositions comprising anti-ICOS antibodies of the IgG1 and/or IgG3human isotype, as well as to compositions comprising anti-ICOSantibodies of the IgG2 and/or IgG4 human isotype that may mediate humanADCC, CDC, and/or antibody-dependent phagocytosis.

The present invention is further directed to methods for the treatmentand prevention of T cell-mediated diseases and disorders, such as, butnot limited to, chronic infection, autoimmune disease or disorder,inflammatory disease or disorder, graft-versus-host disease (GVHD),transplant rejection, and T cell proliferative disorder usingtherapeutic anti-ICOS antibodies with enhanced effector function.

2. BACKGROUND

ICOS is a type I transmembrane protein comprising an extracellular(Ig)V-like domain. ICOS serves as the receptor for the B7hco-stimulatory molecule. ICOS expression is low on naïve human T cellsbut becomes upregulated within hours after TCR engagement. ICOSexpression persists on activated T cells subpopulations such as Th1,Th2, and Th17 CD4⁺ cells.

Given that ICOS expression is concentrated on activated T helper cellpopulations, the therapeutic use of an anti-ICOS antibody with enhancedeffector function holds the promise of improving the efficacy oftreatment and prevention of T cell-mediated diseases and disorders, suchas, but not limited to, chronic infection, autoimmune disease ordisorder, inflammatory disease or disorder, graft-versus-host disease(GVHD), transplant rejection, and T cell proliferative disorder usingtherapeutic anti-ICOS antibodies with enhanced effector function.

3. SUMMARY

The present invention relates to anti-ICOS antibodies with enhancedeffector function that bind to the human ICOS molecule, as well as tocompositions comprising those antibodies. In one embodiment, the presentinvention provides JMab-136 anti-ICOS antibodies (see, U.S. Pat. No.6,803,039) that are able to mediate an antibody effector function moreefficiently than the parental JMab-136 antibody. In one embodiment, ananti-ICOS antibody of the invention comprises a variant Fc region. Inone embodiment, an anti-ICOS antibody of the invention comprises aglycosylation pattern different from that of the parental antibody.

The present invention also provides pharmaceutical compositionscomprising an anti-ICOS antibody with enhanced effector function.

The present invention also relates to methods of treating or preventingT cell-mediated diseases and disorders, such as, but not limited to,chronic infection, autoimmune disease or disorder, inflammatory diseaseor disorder, graft-versus-host disease (GVHD), transplant rejection, andT cell proliferative disorder using therapeutic anti-ICOS antibodieswith enhanced effector function.

3.1. DEFINITIONS

As used herein, the terms “antibody” and “antibodies” (immunoglobulins)encompass monoclonal antibodies (including full-length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, humanantibodies, humanized antibodies, camelised antibodies, chimericantibodies, single-chain Fvs (scFv), single-chain antibodies, singledomain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments,antibody fragments that exhibit the desired biological activity,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies of the invention),intrabodies, and epitope-binding fragments of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Native antibodies are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has at one end a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain at one end (VL) and a constant domain at its other end;the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Light chains areclassified as either lambda chains or kappa chains based on the aminoacid sequence of the light chain constant region. The variable domain ofa kappa light chain may also be denoted herein as VK. The term “variableregion” may also be used to describe the variable domain of a heavychain or light chain. Particular amino acid residues are believed toform an interface between the light and heavy chain variable domains.Such antibodies may be derived from any mammal, including, but notlimited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice,etc.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in segments called Complementarity Determining Regions(CDRs) both in the light chain and the heavy chain variable domains. Themore highly conserved portions of the variable domains are called theframework regions (FW). The variable domains of native heavy and lightchains each comprise four FW regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FW regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see, Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are generally not involved directly in antigen binding, but mayinfluence antigen binding affinity and may exhibit various effectorfunctions, such as participation of the antibody in ADCC, CDC,antibody-dependent phagocytosis and/or apoptosis.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are associated with its binding toantigen. The hypervariable regions encompass the amino acid residues ofthe “complementarity determining regions” or “CDRs” (e.g., residues24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domainand residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, J. Mol. Biol., 196:901-917(1987)). “Framework” or “FW” residues are those variable domain residuesflanking the CDRs. FW residues are present in chimeric, humanized,human, domain antibodies, diabodies, vaccibodies, linear antibodies, andbispecific antibodies.

As used herein “Fc region” includes the polypeptides comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, and the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM Fc may include the J chain.For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Althoughthe boundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat et al. (1991, NIH Publication 91-3242, National TechnicalInformation Service, Springfield, Va.). The “EU index as set forth inKabat” refers to the residue numbering of the human IgG1 EU antibody asdescribed in Kabat et al. supra. Fc may refer to this region inisolation, or this region in the context of an antibody, antibodyfragment, or Fc fusion protein. An Fc variant protein may be anantibody, Fc fusion, or any protein or protein domain that comprises anFc region. Particularly preferred are proteins comprising variant Fcregions, which are non-naturally occurring variants of an Fc region. Theamino acid sequence of a non-naturally occurring Fc region (alsoreferred to herein as a “variant Fc region”) comprises a substitution,insertion and/or deletion of at least one amino acid residue compared tothe wild type amino acid sequence. Any new amino acid residue appearingin the sequence of a variant Fc region as a result of an insertion orsubstitution may be referred to as a non-naturally occurring amino acidresidue. Note: Polymorphisms have been observed at a number of Fcpositions, including but not limited to Kabat 270, 272, 312, 315, 356,and 358, and thus slight differences between the presented sequence andsequences in the prior art may exist.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, monoclonal antibodies areadvantageous in that they can be synthesized by hybridoma cells that areuncontaminated by other immunoglobulin producing cells. Alternativeproduction methods are known to those trained in the art, for example, amonoclonal antibody may be produced by cells stably or transientlytransfected with the heavy and light chain genes encoding the monoclonalantibody.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring engineering of theantibody by any particular method. The term “monoclonal” is used hereinto refer to an antibody that is derived from a clonal population ofcells, including any eukaryotic, prokaryotic, or phage clone, and notthe method by which the antibody was engineered. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., Nature, 256:495 (1975), or may be made by any recombinant DNAmethod (see, e.g., U.S. Pat. No. 4,816,567), including isolation fromphage antibody libraries using the techniques described in Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991), for example. These methods can be used to producemonoclonal mammalian, chimeric, humanized, human, domain antibodies,diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

A “human antibody” can be an antibody derived from a human or anantibody obtained from a transgenic organism that has been “engineered”to produce specific human antibodies in response to antigenic challengeand can be produced by any method known in the art. In certaintechniques, elements of the human heavy and light chain loci areintroduced into strains of the organism derived from embryonic stem celllines that contain targeted disruptions of the endogenous heavy chainand light chain loci. The transgenic organism can synthesize humanantibodies specific for human antigens, and the organism can be used toproduce human antibody-secreting hybridomas. A human antibody can alsobe an antibody wherein the heavy and light chains are encoded by anucleotide sequence derived from one or more sources of human DNA. Afully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, or in vitroactivated ICOS expressing T cells, all of which are known in the art.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells (e.g.,Natural Killer (NK) cells, neutrophils, and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In one embodiment, such cells are human cells. While not wishingto be limited to any particular mechanism of action, these cytotoxiccells that mediate ADCC generally express Fc receptors (FcRs). Theprimary cells for mediating ADCC, NK cells, express FcγRIII, whereasmonocytes express FcγRI, FcγRII, FcγRIII and/or FcγRIV. FcR expressionon hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev.Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an invitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecules of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA),95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to initiate complement activation and lyse a target in thepresence of complement. The complement activation pathway is initiatedby the binding of the first component of the complement system (C1q) toa molecule (e.g., an antibody) complexed with a cognate antigen. Toassess complement activation, a CDC assay, e.g., as described inGazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

“Antibody-dependent phagocytosis” or “opsonization” as used hereinrefers to the cell-mediated reaction wherein nonspecific cytotoxic cellsthat express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. The cells express at least FcγRI, FCγRII,FcγRIII and/or FcγRIV and carry out ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, the FcR is anative sequence human FcR. Moreover, in certain embodiments, the FcR isone which binds an IgG antibody (a gamma receptor) and includesreceptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain. (See, Daëron, Annu. Rev. Immunol.,15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994);and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol.,24:249 (1994)).

“Affinity” of an antibody for an epitope to be used in the treatment(s)described herein is a term well understood in the art and means theextent, or strength, of binding of antibody to epitope. Affinity may bemeasured and/or expressed in a number of ways known in the art,including, but not limited to, equilibrium dissociation constant (KD orKd), apparent equilibrium dissociation constant (KD′ or Kd′), and IC₅₀(amount needed to effect 50% inhibition in a competition assay). It isunderstood that, for purposes of this invention, an affinity is anaverage affinity for a given population of antibodies which bind to anepitope. Values of KD′ reported herein in terms of mg IgG per mL ormg/mL indicate mg Ig per mL of serum, although plasma can be used. Whenantibody affinity is used as a basis for administration of the treatmentmethods described herein, or selection for the treatment methodsdescribed herein, antibody affinity can be measured before and/or duringtreatment, and the values obtained can be used by a clinician inassessing whether a human patient is an appropriate candidate fortreatment.

As used herein, the term “avidity” is a measure of the overall bindingstrength (i.e., both antibody arms) with which an antibody binds anantigen. Antibody avidity can be determined by measuring thedissociation of the antigen-antibody bond in antigen excess using anymeans known in the art, such as, but not limited to, by the modificationof indirect fluorescent antibody as described by Gray et al., J. Virol.Meth., 44:11-24. (1993) An “epitope” is a term well understood in theart and means any chemical moiety that exhibits specific binding to anantibody. An “antigen” is a moiety or molecule that contains an epitope,and, as such, also specifically binds to antibody. The term “antibodyhalf-life” as used herein means a pharmacokinetic property of anantibody that is a measure of the mean survival time of antibodymolecules following their administration. Antibody half-life can beexpressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body or a specificcompartment thereof, for example, as measured in serum or plasma, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The term “isotype” refers to the classification of an antibody's heavyor light chain constant region. The constant domains of antibodies arenot involved in binding to antigen, but exhibit various effectorfunctions. Depending on the amino acid sequence of the heavy chainconstant region, a given human antibody or immunoglobulin can beassigned to one of five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM. Several of these classes may be further divided intosubclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma3), and IgG4 (gamma 4), and IgA1 and IgA2. The heavy chain constantregions that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1 and IgG3 are known to mediate ADCC in humans. Human light chainconstant regions may be classified into two major classes, kappa andlambda

As used herein, the term “immunogenicity” means that a compound iscapable of provoking an immune response (stimulating production ofspecific antibodies and/or proliferation of specific T cells).

As used herein, the term “antigenicity” means that a compound isrecognized by an antibody or may bind to an antibody and induce animmune response.

By the terms “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is an inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness. Thus, the terms “treat,” “treating” or“treatment of” (or grammatically equivalent terms) refer to bothprophylactic and therapeutic treatment regimes.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result refers to an amount of an antibody orcomposition of the invention that is effective to produce a desiredeffect, which is optionally a therapeutic effect (i.e., byadministration of a therapeutically effective amount). For example, a“sufficient amount” or “an amount sufficient to” can be an amount thatis effective to deplete ICOS expressing T cells.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Stated in anotherway, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation, and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence of the VH (A) and VL (B) domains of theJMab-136 anti-ICOS antibody. CDR residues, defined according to Kabat,are in boxed bold letters. Potential O-glycosylation sites (T or Sresidue) and deamidation sites (DS or DG residues) are highlighted ingray.

FIG. 2. Enhanced binding affinity of IC9G1-aFuc to human and cynomolgusFcgRIIIa. Binding affinity (nM) of IC9G1-aFuc to recombinant human andcynomolgus FcγRs was measured as compared to a control antibodies (IC009and IC9G1) and is summarized in this Figure.

FIG. 3. IC9G1-aFuc inhibits CD3/ICOSL induced human T Cellproliferation. Human T cells were incubated for 72 hrs on a plate coatedwith B7h-Fc (50 μl of 4 μg/ml) and anti-CD3 antibody (50 μl of 0.2μg/ml) in the presence of increasing amounts of the IC9G1-aFuc antibody.T cell proliferation as a function of IC9G1-aFuc antibody concentrationis shown. Data obtained from control experiments using the IC009 andIC9G1 antibodies are also shown.

FIG. 4. IC9G1-aFuc does not inhibit anti-CD3/anti-CD28 antibody mediatedproliferation of human tonsillar T cells. Isolated human tonsillar Tcells were incubated for 72 hrs on a plate coated with anti-CD3 and/oranti-CD28 antibodies. Cell proliferation detected in the presence of 10microg/ml of IC9G1 is shown.

FIG. 5. The ADCC activity of IC9G1-aFuc is higher than that of the IC9G1or IC009 antibodies. ADCC activity was measured using stabletranfectants (A) HPB-ALL cells (HPB-ALL h-ICOS) and (B) Jurkat cells(Jurkat h-ICOS) expressing a human ICOS as target cells. The EC50activity of the IC9G1-aFuc and IC9G1 antibodies on HPB-ALL h-ICOS cellswas 138 pM and 648 pM, respectively. The EC50 activity of the IC9G1-aFucand IC9G1 antibodies on transgenic Jurkat h-ICOS cells was 5.7 pM and 61pM, respectively.

FIG. 6. ICOS expression in human tonsil is restricted to CD4+ memoryT_(FH) cells. The anti-ICOS staining pattern of CD4+ CD45RO-CXCR5− naïveT cells and CD4+ CD45RO+CXCR5+ memory T_(FH) is shown.

FIG. 7. The ADCC activity of IC9G1-aFuc is higher than that of the IC9G1or IC009 antibodies. ADCC activity was measured using isolated humantonsillar T cells as target cells. The EC50 activity of the IC9G1-aFucand IC9G1 antibodies was 8.2 pM and 60.4 pM, respectively, in thisassay.

FIG. 8. IC9G1-aFuc mediated ADCC activity on freshly isolated cynomolgussplenic T cell targets. (A) ICOS expression profile of isolatedcynomolgus splenic CD4+ CD45RA+ naïve T cells and CD4+ CD45RA− memory Tcells was determined using flow cytometry. Flow cytometry plots ofstained cells are shown. ICOS expression level of CD4+ CD45RA− memory Tcells is significantly higher than that of the CD4+ CD45RA+naïve Tcells. (B) ADCC cytotoxicity curves of IC009, IC9G1 and IC9G1-aFucantibodies measured using isolated cynomolgus splenic T cells is shown.The ADCC activity of IC9G1-aFuc is higher than that of either the IC009or IC9G1 antibodies. The EC50 activity of the IC9G1-aFuc and IC9G1antibodies was 14.6 pM and 236 pM, respectively, in this assay.

FIG. 9. IC9G1-aFuc mediated ADCC activity on freshly isolated cynomolgusmesenteric lymph node (MLN) T cell targets. (A) ICOS expression profileof isolated cynomolgus MLN CD4+ CD45RA+ naïve T cells and CD4+ CD45RA−activated T cells was determined flow cytometry. Flow cytometry plots ofstained cells are shown. ICOS expression level of CD4+ CD45RA− activatedT cells is significantly higher than that of the CD4+ CD45RA+ naïve Tcells. (B) ADCC cytotoxicity curves of IC009, IC9G1 and IC9G1-aFucantibodies measured using isolated cynomolgus MLN T cells is shown. TheADCC activity of IC9G1-aFuc is higher than that of either the IC009 orIC9G1 antibodies. The EC50 activity of the IC9G1-aFuc and IC9G1antibodies was 17.1 pM and 198 pM, respectively, in this assay.

FIG. 10. IC9G1-aFuc PK profile in cynomolgus monkeys. A single dose of0.1 mg/kg, 1 mg/kg or 10 mg/kg of IC9G1-aFuc antibody was administeredintravenously to cynomolgus monkeys. Serum concentration of theIC9G1-aFuc antibody was measured for 4 weeks post-administration.IC9G1-aFuc serum concentration as a function of time is shown.

FIG. 11. A single IV dose of IC9G1-aFuc significantly depletes the levelof CD3+ CD4+CD45RA_ICOS+ memory T cells in cynomolgus monkeys in vivo. Asingle dose of 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg or 10 mg/kg of IC9G1-aFucantibody was administered intravenously to cynomolgus monkeys. The levelCD3+ CD4+CD45RA-ICOS+ memory T cells was monitored over time. Normalizedmemory T cell levels as a function of time after IC9G1-aFucadministration is shown. Administration of a single dose of 0.1 mg/kg, 1mg/kg or 10 mg/kg of IC9G1-aFuc antibody achieved essentially completeelimination of CD3+CD4+CD45RA_ICOS+ memory T cells by day 4. Therecovery of the ICOS+ memory T cells was dose dependent.

FIG. 12. Flow cytometry based characterization of germinal center Bcells. Cynomolgus germinal center B cells were identified either asCD3-CD20+IgM-CD95+ cells or CD3-CD20+IgM-CD27+ cells.

FIG. 13. A single IV dose of IC9G1-aFuc significantly reduces the levelof mesenteric lymph node (MLN) memory helper ICOS+ T cells and MLNgerminal center B cells in cynomolgus monkeys in vivo. A single dose of0.1 mg/kg, or 10 mg/kg of IC9G1-aFuc antibody was administeredintravenously to cynomolgus monkeys. Control animals were treated with10 mg/kg IC009 or PBS. The level MLN memory ICOS+ T cells and MLNgerminal center B cells were monitored over time. MLN memory helper Tcells were identified as CD3+ CD4+CD45RA-ICOS+ cells. MLN germinalcenter B cells were identified as CD20+CD95+IgM−cells. (A) Total numberof MLN T cells and MLN germinal center B cells on day 8 after treatmentare shown. (B) Percent depletion of ICOS+ T cells and % dissolution ofgerminal center B cells on day 8 after treatment are shown. IC9G1-aFucadministration resulted in a dose dependent depletion of the memoryhelper ICOS+ T cells and germinal center B cells from the MLN.

FIG. 14. A single IV dose of IC9G1-aFuc significantly reduces the levelof splenic memory helper ICOS+ T cells and germinal center B cells incynomolgus monkeys in vivo. A single dose of 0.1 mg/kg, or 10 mg/kg ofIC9G1-aFuc antibody was administered intravenously to cynomolgusmonkeys. Control animals were treated with 10 mg/kg IC009 or PBS. Thelevel of splenic memory ICOS+ T cells and germinal center B cells weremonitored over time. Splenic memory helper T cells were identified asCD3+CD4+CD45RA-ICOS+ cells; germinal center B cells were identified asCD3−CD20+CD95+IgM−cells. (A) Total number of splenic memory helper Tcells and germinal center B cells on days 8 and 30 after treatment areshown. (B) Percent depletion of T cells and % dissolution of germinalcenter B cells on days 8 and 29 after treatment are shown. IC9G1-aFucadministration resulted in a significant depletion of the memory helperT cells and germinal center B cells from the spleen. Depletion levelswere significantly higher in animals receiving IC9G1-aFuc than incontrol animals receiving the IC009 antibody. Maximum depletion of Tcells was achieved by day 8 after IC9G1-aFuc administration. Maximumlevel of germinal center B cell depletion was seen on day 29 afterIC9G1-aFuc administration.

FIG. 15. Splenic germinal centers were atrophied on day 29 afteradministration of a single dose of IC9G1-aFuc antibody to cynomolgusmonkeys. The morphology of splenic white pulp was examined following theadministration of a single dose f IC9G1-aFuc antibody. Histologicalsections of the spleen isolated on day 8 (A) and day 29 (B) afterIC9G1-aFuc antibody administration are shown. IC9G1-aFuc administrationresults in severe atrophy of splenic follicles by day 29.

FIG. 16. Amino acid sequence alignment of long and short isoforms ofhuman ICOS (SEQ ID NO: 32 and 33, respectively).

FIG. 17. Nucleotide sequence complementarity of ICOS mRNA (SEQ ID NO:34)and selected micro RNA molecules.

FIG. 18. Relative expression level of miR-101 in muscle specimen frominclusion-body myositis (IBM), polymyositis (PM), and dermatomyositis(DM) patients compared to healthy normal controls as measured by TaqManQRT-PCR.

FIG. 19. Relative levels of (A) ICOS and ICOS-L, (B) CD4 and (C) CD3εmRNA in muscle specimen from inclusion-body myositis (IBM), polymyositis(PM), and dermatomyositis (DM) patients compared with normal controls asmeasured by Affymetrix whole genome array. (D) ICOS and ICOS-L mRNAexpression levels in whole blood (WB) samples isolated frominclusion-body myositis (IBM), polymyositis (PM), and dermatomyositis(DM) patients compared to normal controls as measured by TaqMan QRT-PCR.

FIG. 20. Relative mRNA expression levels of (A) ICOS and ICOSL, (B) CD4and (C) CD3ε in affected skin lesions of SLE patients compared to normalcontrols as measured by TaqMan QRT-PCR.

FIG. 21. Relative mRNA expression levels of (A) CD28, CTLA4, ICOS,ICOS-L, (B) CD4 and (C) CD38 in whole blood (WB) from SLE patientscompared to normal controls as measured by TaqMan QRT-PCR.

5. DETAILED DESCRIPTION

The present invention relates to methods for generating anti-ICOSantibodies with enhanced effector function. Using the methods of theinvention, an anti-ICOS parental antibody is modified to yield ananti-ICOS antibody with enhanced effector function, such as, but notlimited to, enhanced ADCC, enhanced CDC, and enhanced antibody-dependentphagocytosis. Any anti-ICOS antibody that specifically binds to thehuman ICOS antigen may serve as a parental antibody for the purpose ofpracticing a method of the present invention. In one embodiment,anti-ICOS antibodies disclosed in U.S. Pat. No. 6,803,039 serve asparental antibody. In a specific embodiment, the JMAb-136 (IgG2)anti-ICOS antibody serves as the parental antibody.

The present invention provides anti-ICOS antibodies with enhancedeffector function. In one embodiment, an anti-ICOS antibody of theinvention mediates an antibody dependent effector function moreefficiently than the parental anti-ICOS antibody. In a specificembodiment, an anti-ICOS antibody of the invention mediates an antibodydependent effector function more efficiently than the JMAb-136 (see,U.S. Pat. No. 6,803,039).

In one embodiment, an anti-ICOS antibody described herein mediates anantibody dependent effector function more efficiently than the parentalanti-ICOS antibody wherein said effector function is selected from thegroup consisting of: antibody-dependent cell-mediated cytotoxicity(ADCC), complement mediated cytotoxicity (CDC), antibody-dependentphagocytosis. In one embodiment, an anti-ICOS antibody of the inventionmediates antibody-dependent cell-mediated cytotoxicity (ADCC) moreefficiently than the parental anti-ICOS antibody. In another embodiment,an anti-ICOS antibody of the invention mediates complement mediatedcytotoxicity (CDC) more efficiently than the parental anti-ICOSantibody. In a further embodiment, an anti-ICOS antibody of theinvention mediates antibody-dependent phagocytosis more efficiently thanthe parental anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) more efficientlythan the parental anti-ICOS antibody wherein the ADCC activity isdetermined using an in vitro cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vitro ADCC assay than theparental anti-ICOS antibody. In another specific embodiment, ananti-ICOS antibody of the invention mediates at least 2 fold, at least 3fold, at least 4 fold, at least 5 fold or at least 10 fold highermaximum cytotoxicity in an in vitro ADCC assay than the parentalanti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) more efficientlythan the JMab-136 anti-ICOS antibody wherein the ADCC activity isdetermined using an in vitro cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vitro ADCC assay than theJMab-136 anti-ICOS antibody. In another specific embodiment, ananti-ICOS antibody of the invention mediates at least 2 fold, at least 3fold, at least 4 fold, at least 5 fold or at least 10 fold highermaximum cytotoxicity in an in vitro ADCC assay than the JMab-136anti-ICOS antibody.

In one embodiment, the EC50 of an anti-ICOS antibody of the invention inan in vitro ADCC assay is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody.

In another embodiment, the EC50 of an anti-ICOS antibody of theinvention in an in vitro ADCC assay is at least about 2×, at least about5×, at least about 10×, at least about 20×, at least about 50×, or atleast about 100× lower than that of the JMab-136 anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) more efficientlythan the parental anti-ICOS antibody wherein the ADCC activity isdetermined using an in vivo cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vivo ADCC assay than theparental anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) more efficientlythan the JMab-136 anti-ICOS antibody wherein the ADCC activity isdetermined using an in vivo cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vivo ADCC assay than theJMab-136 anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatescomplement-dependent cytotoxicity (CDC) more efficiently than theparental anti-ICOS antibody wherein the CDC activity is determined usingan in vitro cytotoxicity assay. In a specific embodiment, an anti-ICOSantibody of the invention mediates at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 75%, at least about 100% higher maximumcytotoxicity in an in vitro CDC assay than the parental anti-ICOSantibody.

In one embodiment, an anti-ICOS antibody of the invention mediatescomplement-dependent cytotoxicity (CDC) more efficiently than theJMab-136 anti-ICOS antibody wherein the CDC activity is determined usingan in vitro cytotoxicity assay. In a specific embodiment, an anti-ICOSantibody of the invention mediates at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 75%, at least about 100% higher maximumcytotoxicity in an in vitro CDC assay than the JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 of an anti-ICOS antibody of the invention inan in vitro CDC assay is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody. In anotherembodiment, the EC50 of an anti-ICOS antibody of the invention in an invitro CDC assay is at least about 2×, at least about 5×, at least about10×, at least about 20×, at least about 50×, or at least about 100×lower than that of the JMab-136 anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent phagocytosis more efficiently than the parentalanti-ICOS antibody wherein the antibody-dependent phagocytosis activityis determined using an in vitro cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vitro antibody-dependentphagocytosis assay than the parental anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention mediatesantibody-dependent phagocytosis more efficiently than the JMab-136anti-ICOS antibody wherein the antibody-dependent phagocytosis activityis determined using an in vitro cytotoxicity assay. In a specificembodiment, an anti-ICOS antibody of the invention mediates at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 75%, at least about100% higher maximum cytotoxicity in an in vitro antibody-dependentphagocytosis assay than the JMab-136 anti-ICOS antibody.

In one embodiment, the EC50 of an anti-ICOS antibody of the invention inan in vitro antibody-dependent phagocytosis assay is at least about 2×,at least about 5×, at least about 10×, at least about 20×, at leastabout 50×, or at least about 100× lower than that of the parentalanti-ICOS antibody. In another embodiment, the EC50 of an anti-ICOSantibody of the invention in an in vitro antibody-dependent phagocytosisassay is at least about 2×, at least about 5×, at least about 10×, atleast about 20×, at least about 50×, or at least about 100× lower thanthat of the JMab-136 anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region. In another embodiment, an anti-ICOS antibody of theinvention comprises a variant Fc region that has an altered affinity foran Fc ligand protein. In a further embodiment, an anti-ICOS antibody ofthe invention comprises a variant Fc region that has an altered affinityfor an Fc ligand selected from the group consisting of: FcγRIA, FcγRIIA,FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, and C1q. In a specific embodiment,an anti-ICOS antibody of the invention comprises a variant Fc regionthat has an altered affinity for the FcγRIIIA protein. In a furtherembodiment, an anti-ICOS antibody of the invention comprises a variantFc region that has an altered affinity for the C1q protein. In aspecific embodiment, an Fc ligand protein may be a mouse, human orprimate (e.g., cynomolgus) Fc ligand protein.

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region that has an increased affinity for an Fc ligandprotein. In a further embodiment, an anti-ICOS antibody of the inventioncomprises a variant Fc region that has an increased affinity for an Fcligand selected from the group consisting of: FcγRIA, FcγRIIA, FcγRIIB,FcγRIIIA, FcγRIIIB, FcγRIV, and C1q. In a specific embodiment, ananti-ICOS antibody of the invention comprises a variant Fc region thathas an increased affinity for the FcγRIIIA protein. In a furtherembodiment, an anti-ICOS antibody of the invention comprises a variantFc region that has an increased affinity for the C1q protein. In aspecific embodiment, an Fc ligand protein may be a mouse, human orprimate (e.g., cynomolgus) Fc ligand protein.

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region wherein said variant Fc region comprises at least oneamino acid substitution, insertion or deletion. In another embodiment,an anti-ICOS antibody of the invention comprises a variant Fc regioncomprising at least one amino acid substitution, insertion or deletionwherein said at least one amino acid residue substitution, insertion ordeletion results in an increased affinity for an Fc ligand selected fromthe group consisting of: FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB,FcγRIV, and C1q. In a specific embodiment, an anti-ICOS antibody of theinvention comprises a variant Fc region comprising at least one aminoacid substitution, insertion or deletion wherein said at least one aminoacid residue substitution, insertion or deletion results in an increasedaffinity for the FcγRIIIA protein. In a further embodiment, an anti-ICOSantibody of the invention comprises a variant Fc region comprising atleast one amino acid substitution, insertion or deletion wherein said atleast one amino acid residue substitution, insertion or deletion resultsin an increased affinity for the C1q protein. In a specific embodiment,an Fc ligand protein may be a mouse, human or primate (e.g., cynomolgus)Fc ligand protein.

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region comprising at least one amino acid substitution,insertion or deletion wherein said at least one amino acid residue isselected from the group consisting of: residue 239, 330, and 332,wherein amino acid residues are numbered following the EU index. Inanother embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region comprising at least one amino acid substitution,insertion or deletion wherein said at least one substituted, inserted ordeleted amino acid residue is selected from the group consisting of:residue 239, 330, and 332, wherein amino acid residues are numberedfollowing the EU index. In a further embodiment, an anti-ICOS antibodydescribed herein comprises a variant Fc region comprising at least oneamino acid substitution wherein said at least one substituted amino acidresidue is selected from the group consisting of: residue 239, 330, and332, wherein amino acid residues are numbered following the EU index. Inanother embodiment, an anti-ICOS antibody described herein comprises avariant Fc region comprising at least one amino acid substitutionwherein said at least one amino acid substitution is selected from thegroup consisting of: S239D, A330L, A330Y, and 1332E, wherein amino acidresidues are numbered following the EU index. In a specific embodiment,an anti-ICOS antibody of the invention comprises a variant Fc regioncomprising the S239D, A330L, and I332E amino acid substitutions, whereinamino acid residues are numbered following the EU index.

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region comprising at least one of the amino acid residuesselected from the group consisting of: D at residue 239, E at residue239, L at residue 330, Y at residue 330, E at residue 332, and D atresidue 332, wherein amino acid residues are numbered following the EUindex. In a specific embodiment, an anti-ICOS antibody of the inventioncomprises a variant Fc region comprising D at residue 239, L at residue330, and E at residue 332, wherein amino acid residues are numberedfollowing the EU index.

In one embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region wherein the engineered Fc region comprises aposttranslational modification that is different from that of theparental anti-ICOS antibody. In a specific embodiment, an anti-ICOSantibody of the invention comprises an engineered Fc region wherein saidengineered Fc region comprises complex N-glycoside-linked sugar chainsin which fucose is not bound to N-acetylglucosamine in the reducing endin the sugar chain.

In one embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region that has an altered affinity for an Fc ligandprotein. In a further embodiment, an anti-ICOS antibody of the inventioncomprises an engineered Fc region that has an altered affinity for an Fcligand selected from the group consisting of: FcγRIA, FcγRIIA, FcγRIIB,FcγRIIIA, FcγRIIIB, FcγRIV, and C1q. In a specific embodiment, ananti-ICOS antibody of the invention comprises an engineered Fc regionthat has an altered affinity for the FcγRIIIA protein. In a furtherembodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region that has an altered affinity for the C1q protein.

In one embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region that has an increased affinity for an Fc ligandprotein. In a further embodiment, an anti-ICOS antibody of the inventioncomprises an engineered Fc region that has an increased affinity for anFc ligand selected from the group consisting of: FcγRIA, FcγRIIA,FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, and C1q. In a specific embodiment,an anti-ICOS antibody of the invention comprises an engineered Fc regionthat has an increased affinity for the FcγRIIIA protein. In a furtherembodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region that has an increased affinity for the C1q protein.

In one embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region wherein said engineered Fc region comprises areduced level of fucose compared to a native antibody. In anotherembodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region comprising a reduced level of fucose, wherein saidreduction in fucose level results in an increased affinity for an Fcligand selected from the group consisting of: FcγRIA, FcγRIIA, FcγRIIB,FcγRIIIA, FcγRIIIB, FcγRIV, and C1q. In a specific embodiment, ananti-ICOS antibody of the invention comprises an engineered Fc regioncomprising a reduced level of fucose, wherein said reduction in fucoselevel results in an increased affinity for the FcγRIIIA protein. In afurther embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region comprising a reduced level of fucose, wherein saidreduction in fucose level results in an increased affinity for the C1qprotein.

Anti-ICOS antibodies described herein comprise Fc regions having a highbinding affinity for the human FcγRIIIA protein. In one embodiment, ananti-ICOS antibody of the invention comprises an Fc region that has anaffinity constant or K_(a) (k_(on)/k_(off)) of at least 10³ M⁻¹, atleast 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹,at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10³ M⁻¹, at least10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, or at least5×10¹² M⁻¹. In another embodiment, an anti-ICOS antibody of theinvention comprises an Fc region that has a dissociation constant orK_(d) (k_(off)/k_(on)) of less than 5×10⁻³ M, less than 10⁻³ M, lessthan 5×10⁻⁴ M, less than 10⁻⁴ M, less than 5×10⁻⁵ M, less than 10⁻⁵ M,less than 5×10⁻⁶ M, less than 10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷M, less than 5×10⁻⁸ M, less than 10⁻⁸ M, less than 5×10⁻⁹ M, less than10⁻⁹ M, less than 5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than 5×10¹¹ M⁻¹,less than 10⁻¹¹ M, less than 5×10⁻¹² M, or less than 10⁻¹² M.

An antibody used in accordance with a method described herein maycomprise an Fc region that binds to human FcγRIIIA with a dissociationconstant (K_(d)) of less than 3000 nM, less than 2500 nM, less than 2000nM, less than 1500 nM, less than 1000 nM, less than 750 nM, less than500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10nM, less than 5 nM, less than 1 nM as assessed using a method describedherein or known to one of skill in the art (e.g., a BIAcore assay,ELISA) (Biacore International AB, Uppsala, Sweden). In a specificembodiment, an antibody used in accordance with a method describedherein may comprise an Fc region that binds to human FcγRIIIA with adissociation constant (K_(d)) of between 1 to 3000 nM, 1 to 3000 nM, 1to 2000 nM, 1 to 1500 nM, 1 to 1000 nM, 1 to 750 nM, 1 to 500 nM, 1 to250 nM, 1 to 100 nM, 1 to 50 nM, 1 to 25 nM, 1 to 10 nM as assessedusing a method described herein or known to one of skill in the art(e.g., a BIAcore assay, ELISA). In another embodiment, an anti-ICOSantibody used in accordance with a method described herein may comprisean Fc region that binds to human FcγRIIIA with a dissociation constant(K_(d)) of 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM or 1 nM asassessed using a method described herein or known to one of skill in theart (e.g., a BIAcore assay, ELISA).

Anti-ICOS antibodies described herein comprise Fc regions having a highbinding affinity for the non-human primate (e.g., cynomolgus) FcγRIIIAprotein. In one embodiment, an anti-ICOS antibody of the inventioncomprises an Fc region that has an affinity constant or K_(a)(k_(on)/k_(off)) of at least 10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁴M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵ M⁻¹, at least10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 5×10⁷M⁻¹, atleast 10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹,at least 1010 M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least5×10¹¹ M⁻¹, at least 10¹² M⁻¹, or at least 5×10¹² M⁻¹. In anotherembodiment, an anti-ICOS antibody of the invention comprises an Fcregion that has a dissociation constant or K_(d) (k_(off)/k_(on)) ofless than 5×10⁻³ M, less than 10⁻³ M, less than 5×10⁻⁴ M, less than 10⁻⁴M, less than 5×10⁻⁵ M, less than 10⁻⁵ M, less than 5×10⁻⁶ M, less than10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷ M, less than 5×10⁻⁸ M, lessthan 10⁻⁸ M, less than 5×10⁻⁹ M, less than 10⁻⁹ M, less than 5×10⁻¹⁰ M,less than 10⁻¹⁰ M, less than 5×10⁻¹¹ M, less than 10⁻¹¹ M, less than5×10⁻¹² M, or less than 10⁻¹² M.

An antibody used in accordance with a method described herein maycomprise an Fc region that binds to non-human primate (e.g., cynomolgus)FcγRIIIA with a dissociation constant (K_(d)) of less than 3000 nM, lessthan 2500 nM, less than 2000 nM, less than 1500 nM, less than 1000 nM,less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM,less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM,less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM asassessed using a method described herein or known to one of skill in theart (e.g., a BIAcore assay, ELISA) (Biacore International AB, Uppsala,Sweden). In a specific embodiment, an antibody used in accordance with amethod described herein may comprise an Fc region that binds tonon-human primate (e.g., cynomolgus) FcγRIIIA with a dissociationconstant (K_(d)) of between 1 to 3000 nM, 1 to 3000 nM, 1 to 2000 nM, 1to 1500 nM, 1 to 1000 nM, 1 to 750 nM, 1 to 500 nM, 1 to 250 nM, 1 to100 nM, 1 to 50 nM, 1 to 25 nM, 1 to 10 nM as assessed using a methoddescribed herein or known to one of skill in the art (e.g., a BIAcoreassay, ELISA). In another embodiment, an anti-ICOS antibody used inaccordance with a method described herein may comprise an Fc region thatbinds to non-human primate (e.g., cynomolgus) FcγRIIIA with adissociation constant (K_(d)) of 500 nM, 250 nM, 100 nM, 75 nM, 50 nM,25 nM, 10 nM or 1 nM as assessed using a method described herein orknown to one of skill in the art (e.g., a BIAcore assay, ELISA).

Anti-ICOS antibodies described herein comprise Fc regions having a highbinding affinity for the mouse FcγRIIIA protein. In one embodiment, ananti-ICOS antibody of the invention comprises an Fc region that has anaffinity constant or K_(a) (k_(on)/k_(off)) of at least 10³ M⁻¹, atleast 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least 5×10⁴ M⁻¹, at least 10⁵ M⁻¹,at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 5×10⁶ M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10³ M⁻¹, at least10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, at least 10⁻¹¹ M⁻¹, or at least5×10¹² M⁻¹. In another embodiment, an anti-ICOS antibody of theinvention comprises an Fc region that has a dissociation constant orK_(d) (k_(off)/k_(on)) of less than 5×10⁻³ M, less than 10⁻³ M, lessthan 5×10⁻⁴ M, less than 10⁻⁴ M, less than 5×10⁻⁵ M, less than 10⁻⁵ M,less than 5×10⁻⁶ M, less than 10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷M, less than 5×10⁻⁸ M, less than 10⁻⁸ M, less than 5×10⁻⁹ M, less than10⁻⁹ M, less than 5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than 5×10¹¹ M, lessthan 10⁻¹¹ M, less than 5×10⁻¹² M, or less than 10⁻¹² M.

An antibody used in accordance with a method described herein maycomprise an Fc region that binds to mouse FcγRIIIA with a dissociationconstant (K_(d)) of less than 3000 nM, less than 2500 nM, less than 2000nM, less than 1500 nM, less than 1000 nM, less than 750 nM, less than500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10nM, less than 5 nM, less than 1 nM as assessed using a method describedherein or known to one of skill in the art (e.g., a BIAcore assay,ELISA) (Biacore International AB, Uppsala, Sweden). In a specificembodiment, an antibody used in accordance with a method describedherein may comprise an Fc region that binds to mouse FcγRIIIA with adissociation constant (K_(d)) of between 1 to 3000 nM, 1 to 3000 nM, 1to 2000 nM, 1 to 1500 nM, 1 to 1000 nM, 1 to 750 nM, 1 to 500 nM, 1 to250 nM, 1 to 100 nM, 1 to 50 nM, 1 to 25 nM, 1 to 10 nM as assessedusing a method described herein or known to one of skill in the art(e.g., a BIAcore assay, ELISA). In another embodiment, an anti-ICOSantibody used in accordance with a method described herein may comprisean Fc region that binds to mouse FcγRIIIA with a dissociation constant(K_(d)) of 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 10 nM or 1 nM asassessed using a method described herein or known to one of skill in theart (e.g., a BIAcore assay, ELISA).

In one embodiment, anti-ICOS antibodies of the invention comprise one,two, three, four, five, or all six of the CDRs of JMAb-136 (see, U.S.Pat. No. 6,803,039).

The amino acid sequences for CDR1, CDR2, and CDR3 of the heavy chainvariable region of JMAb-136 defined according to Kabat are identified asSEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. The amino acidsequences for CDR1, CDR2 and CDR3 of the light chain variable region ofJMAb-136 defined according to Kabat are identified as SEQ ID NO:3, SEQID NO:4, and SEQ ID NO:5, respectively.

Kabat numbering is based on the seminal work of Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Publication No.91-3242, published as a three volume set by the National Institutes ofHealth, National Technical Information Service (hereinafter “Kabat”).Kabat provides multiple sequence alignments of immunoglobulin chainsfrom numerous species antibody isotypes. The aligned sequences arenumbered according to a single numbering system, the Kabat numberingsystem. The Kabat sequences have been updated since the 1991 publicationand are available as an electronic sequence database (latestdownloadable version 1997). Any immunoglobulin sequence can be numberedaccording to Kabat by performing an alignment with the Kabat referencesequence. Accordingly, the Kabat numbering system provides a uniformsystem for numbering immunoglobulin chains. Unless indicated otherwise,all immunoglobulin amino acid sequences described herein are numberedaccording to the Kabat numbering system. Similarly, all single aminoacid positions referred to herein are numbered according to the Kabatnumbering system. In certain embodiments, an anti-ICOS antibodydescribed herein may comprise a heavy chain variable region, VH,comprising at least one CDR having the amino acid sequence selected fromthe group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10. Incertain embodiments, an anti-ICOS antibody of the invention may comprisea VH domain having the amino acid sequence of SEQ ID NO:7.

In certain embodiments, an anti-ICOS antibody described herein maycomprise a light chain variable region, VK, comprising at least one CDRhaving an amino acid sequence selected from the group consisting of SEQID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In certain embodiments, ananti-ICOS antibody of the invention may comprise a VK domain having theamino acid sequence of SEQ ID NO:2.

In one embodiment, an anti-ICOS antibody of the invention comprises a VKdomain having the amino acid sequence of SEQ ID NO:2 and furthercomprises a VH domain having the amino acid sequence of SEQ ID NO:7.

The present invention encompasses antibodies that bind to human ICOS,comprising derivatives of the VH domain, VH CDR1, VH CDR2, VH CDR3, VKdomain, VK CDR1, VK CDR2, or VK CDR3 described herein that may bind tohuman ICOS. Standard techniques known to those of skill in the art canbe used to introduce mutations (e.g., additions, deletions, and/orsubstitutions) in the nucleotide sequence encoding an antibody,including, for example, site-directed mutagenesis and PCR-mediatedmutagenesis that are routinely used to generate amino acidsubstitutions. In one embodiment, the VH and/or VK CDR derivatives mayinclude less than 25 amino acid substitutions, less than 20 amino acidsubstitutions, less than 15 amino acid substitutions, less than 10 aminoacid substitutions, less than 5 amino acid substitutions, less than 4amino acid substitutions, less than 3 amino acid substitutions, lessthan 2 amino acid substitutions, or 1 amino acid substitution relativeto the original VH and/or VK CDRs of the JMab-136 anti-ICOS antibody. Inanother embodiment, the VH and/or VK CDR derivatives may haveconservative amino acid substitutions (e.g. supra) made at one or morepredicted non-essential amino acid residues (i.e., amino acid residueswhich are not critical for the antibody to specifically bind to humanICOS). Mutations can also be introduced randomly along all or part ofthe VH and/or VK CDR coding sequences, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity. Followingmutagenesis, the encoded antibody can be expressed and the activity ofthe antibody can be determined.

The present invention further encompasses antibodies that bind to humanICOS, said antibodies or antibody fragments comprising one or more CDRswherein said CDRs comprise an amino acid sequence that is at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of one or more CDRs ofthe JMab-136 anti-ICOS antibody. The percent identity of two amino acidsequences can be determined by any method known to one skilled in theart, including, but not limited to, BLAST protein searches.

The present invention further encompasses antibodies that bind to humanICOS, said antibodies or antibody fragments comprising a VH and/or a VKdomain wherein said VH and/or VK domains comprise an amino acid sequencethat is at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% identical to the amino acid sequenceof the VH and VK domain of the JMab-136 anti-ICOS antibody. The percentidentity of two amino acid sequences can be determined by any methodknown to one skilled in the art, including, but not limited to, BLASTprotein searches.

In one embodiment, an anti-ICOS antibody of the invention may bind tohuman ICOS with an affinity comparable to that of the JMab-136 anti-ICOSantibody.

In one embodiment, an anti-ICOS antibody of the invention specificallybinds the same epitope of ICOS as the JMab-136 anti-ICOS antibody.

In one embodiment, an anti-ICOS antibody specifically competes theJMab-136 anti-ICOS antibody for ICOS binding. The competition assay maybe performed using any binding assay known in the art, for example, butnot limited to ELISA assay, radioimmunoassay, flow cytometry.

The invention further provides polynucleotides comprising a nucleotidesequence encoding an anti-ICOS antibody with enhanced effector function.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, as definedherein, to polynucleotides that encode an anti-ICOS antibody withenhanced effector function.

In one embodiment, a polynucleotide of the invention encoding aneffector function enhanced anti-ICOS antibody described herein comprisesan optimized polynucleotide sequence. In a specific embodiment, apolynucleotide of the invention encoding the VH domain of an antibodydescribed herein comprises the nucleotide sequence of SEQ ID NO: 28. Ina specific embodiment, a polynucleotide of the invention encoding the VKdomain of an antibody described herein comprises the nucleotide sequenceof SEQ ID NO: 29. In a specific embodiment, a polynucleotide of theinvention encoding the heavy chain of an antibody described hereincomprises the nucleotide sequence of SEQ ID NO: 30. In a specificembodiment, a polynucleotide of the invention encoding the light chainof an antibody described herein comprises the nucleotide sequence of SEQID NO: 31.

Another embodiment of the invention is a vector comprising one or morenucleotide sequences encoding an anti-ICOS antibody with enhancedeffector function.

In one embodiment, a vector of the invention comprises one or morenucleotide sequences encoding an anti-ICOS antibody with enhancedeffector function wherein the nucleotide sequence is an optimizednucleotide sequence. In a specific embodiment, a vector of the inventioncomprises the nucleotide sequence of SEQ ID NO: 28. In a specificembodiment, a vector of the invention comprises the nucleotide sequenceof SEQ ID NO: 29. In a specific embodiment, a vector of the inventioncomprises the nucleotide sequence of SEQ ID NO: 30. In a specificembodiment, a vector of the invention comprises the nucleotide sequenceof SEQ ID NO: 31. In a further specific embodiment, a vector of theinvention comprises one or more nucleotide sequences encoding ananti-ICOS antibody with enhanced effector function wherein thenucleotide sequence is selected from the group comprising SEQ IDNO:28-31. In a further specific embodiment, a vector of the inventioncomprises one or more nucleotide sequences encoding an anti-ICOSantibody with enhanced effector function wherein the nucleotide sequenceis selected from the group consisting of SEQ ID NO:28-31.

The present invention further relates to an isolated cell comprising avector wherein said vector comprises one or more nucleotide sequencesencoding an anti-ICOS antibody with enhanced effector function. In aspecific embodiment, an isolated cell of the invention comprises apolynucleotide comprising the nucleotide sequence selected from thegroup comprising SEQ ID NO:28-31. In a further specific embodiment, anisolated cell of the invention comprises a polynucleotide comprising thenucleotide sequence selected from the group consisting of SEQ IDNO:28-31.

Anti-ICOS antibodies of the invention include those of the IgG1, IgG2,IgG3, or IgG4 human isotype.

The present invention further relates to pharmaceutical compositionscomprising an anti-ICOS antibody with enhanced effector function.

In still another other aspect, the present invention is directed towardmethods of treating and preventing T cell-mediated diseases anddisorders, such as, but not limited to, chronic infection, autoimmunedisease or disorder, inflammatory disease or disorder, graft-versus-hostdisease (GVHD), transplant rejection, and T cell proliferative disorder,comprising administering to a human in need thereof atherapeutically-effective amount of a an anti-ICOS antibody withenhanced effector function.

The present invention relates to anti-ICOS antibodies with enhancedeffector function, as well as to compositions comprising thoseantibodies. In certain embodiments, an anti-ICOS antibody of theinvention may mediate antigen-dependent-cell-mediated-cytotoxicity(ADCC). In other embodiments, the present invention is directed towardcompositions comprising an anti-ICOS antibody of the IgG1 and/or IgG3human isotype, as well as to an anti-ICOS antibody of the IgG2 and/orIgG4 human isotype, that may mediate human ADCC, CDC, and/orantibody-dependent phagocytosis.

Anti-ICOS antibodies described herein may have a high binding affinityfor the human ICOS antigen. For example, an antibody described hereinmay have an association rate constant or k_(on) rate (antibody(Ab)+antigen (Ag)^(k-on)→Ab-Ag) of at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least 10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹,at least 5×10⁷ M⁻¹s⁻¹, or at least 10³ M⁻¹s⁻¹.

In another embodiment, an anti-ICOS antibody may have a k_(off) rate((Ab-Ag)^(k-off)→antibody (Ab)+antigen (Ag)) of less than 5×10⁻¹s⁻¹,less than 10⁻¹ s⁻¹, less than 5×10⁻² s⁻¹, less than 10⁻² s⁻¹, less than5×10⁻³ s⁻¹, less than 10⁻³ s⁻¹, less than 5×10⁻⁴ s⁻¹, or less than 10⁻⁴s⁻¹. In a another embodiment, an antibody of the invention has a k_(off)of less than 5×10⁻⁵ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁶ s⁻¹, lessthan 10⁻⁶ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁷ s⁻¹, less than5×10⁻⁸ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁹ s⁻¹, less than 10⁻⁹s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, an anti-ICOS antibody may have an affinityconstant or K_(a) (k_(on)/k_(off)) of at least 10² M⁻¹, at least 5×10²M⁻¹, at least 10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, atleast 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 5×10⁷ M⁻¹, at least 10⁸ M⁻¹,at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 1010M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, atleast 10¹² M⁻¹, at least 5×10¹² M⁻¹, at least 10¹³ M⁻¹, at least 5×10¹³M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 1015 M⁻¹, or atleast 5×10¹⁵ M⁻¹. In yet another embodiment, an anti-ICOS antibody mayhave a dissociation constant or K_(d) (k_(off)/k_(on)) of less than5×10⁻² M, less than 10⁻² M, less than 5×10⁻³ M, less than 10⁻³ M, lessthan 5×10⁻⁴ M, less than 10⁻⁴ M, less than 5×10⁻⁵ M, less than 10⁻⁵ M,less than 5×10⁻⁶ M, less than 10⁻⁶ M, less than 5×10⁻⁷ M, less than 10⁻⁷M, less than 5×10⁻⁸ M, less than 10⁻⁸ M, less than 5×10⁻⁹ M, less than10⁻⁹ M, less than 5×10⁻¹⁰ M, less than 10⁻¹⁰ M, less than 5×10¹¹ M, lessthan 10⁻¹¹ M, less than 5×10⁻¹² M, less than 10⁻¹² M, less than 5×10⁻¹³M, less than 10⁻¹³ M, less than 5×10⁻¹⁴ M, less than 10⁻¹⁴ M, less than5×10⁻¹⁵ M, or less than 10⁻¹⁵ M.

An antibody used in accordance with a method described herein mayimmunospecifically bind to ICOS and may have a dissociation constant(K_(d)) of less than 3000 pM, less than 2500 pM, less than 2000 pM, lessthan 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM,less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM,less than 75 pM as assessed using a method described herein or known toone of skill in the art (e.g., a BIAcore assay, ELISA) (BiacoreInternational AB, Uppsala, Sweden). In a specific embodiment, anantibody used in accordance with a method described herein mayimmunospecifically bind to a human ICOS antigen and may have adissociation constant (K_(d)) of between 25 to 3400 pM, 25 to 3000 pM,25 to 2500 pM, 25 to 2000 pM, 25 to 1500 pM, 25 to 1000 pM, 25 to 750pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, 25 to 75 pM, 25 to 50 pMas assessed using a method described herein or known to one of skill inthe art (e.g., a BIAcore assay, ELISA). In another embodiment, ananti-ICOS antibody used in accordance with a method described herein mayimmunospecifically bind to ICOS and may have a dissociation constant(K_(d)) of 500 pM, 100 pM, 75 pM or 50 pM as assessed using a methoddescribed herein or known to one of skill in the art (e.g., a BIAcoreassay, ELISA).

The invention further provides polynucleotides comprising a nucleotidesequence encoding an anti-ICOS antibody with enhanced effector function.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedherein, to polynucleotides that encode an anti-ICOS antibody withenhanced effector function.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1×SSC/0.2% SDS at about 60° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

A polynucleotide encoding an antibody may also be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the immunoglobulinmay be chemically synthesized or obtained from a suitable source (e.g.,an antibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

The present invention further provides for antibodies that efficientlydeplete ICOS expressing cells in a mouse xenograft model system. In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of ICOS expressing cells in a mouse xenograft modelsystem.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing cells in amouse xenograft model more efficiently than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention depletesICOS expressing cells in a mouse xenograft model more efficiently thanthat of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing cells in a mouse xenograft modelthan that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In one embodiment, administrationof one or more therapeutic doses of an anti-ICOS antibody of theinvention achieves at least about 2-fold, at least about 3-fold, atleast about 5-fold, or at least about 10-fold higher depletion of ICOSexpressing cells in a mouse xenograft model than that of the fucosylatedJMab-136 anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing cells in a mousexenograft model is at least about 2×, at least about 5×, at least about10×, at least about 20×, at least about 50×, or at least about 100×lower than that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment, the EC50value of an anti-ICOS antibody of the invention for the depletion ofICOS expressing cells in a mouse xenograft model is at least about 2×,at least about 5×, at least about 10×, at least about 20×, at leastabout 50×, or at least about 100× lower than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing cells in a mouse xenograft model than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about75%, at least about 100% higher depletion of ICOS expressing cells in amouse xenograft model than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing cells in a mouse xenograft model than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 2×, at least about 5×, at least about 10×, atleast about 15×, at least about 20×, at least about 25×, at least about50×, or at least about 100× higher depletion of ICOS expressing cells ina mouse xenograft model than that of the fucosylated JMab-136 anti-ICOSantibody.

The present invention further provides for antibodies that efficientlydeplete ICOS expressing cells in a transgenic mouse model system. In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of ICOS expressing cells in a transgenic mousemodel system.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing cells in atransgenic mouse model more efficiently than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention depletesICOS expressing cells in a transgenic mouse model more efficiently thanthat of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing cells in a transgenic mouse modelthan that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In one embodiment, administrationof one or more therapeutic doses of an anti-ICOS antibody of theinvention achieves at least about 2-fold, at least about 3-fold, atleast about 5-fold, or at least about 10-fold higher depletion of ICOSexpressing cells in a transgenic mouse model than that of thefucosylated JMab-136 anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing cells in a transgenicmouse model is at least about 2×, at least about 5×, at least about 10×,at least about 20×, at least about 50×, or at least about 100× lowerthan that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment, the EC50value of an anti-ICOS antibody of the invention for the depletion ofICOS expressing cells in a transgenic mouse model is at least about 2×,at least about 5×, at least about 10×, at least about 20×, at leastabout 50×, or at least about 100× lower than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing cells in a transgenic mouse model than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about75%, at least about 100% higher depletion of ICOS expressing cells in atransgenic mouse model than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing cells in a transgenic mouse model than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 2×, at least about 5×, at least about 10×, atleast about 15×, at least about 20×, at least about 25×, at least about50×, or at least about 100× higher depletion of ICOS expressing cells ina transgenic mouse model than that of the fucosylated JMab-136 anti-ICOSantibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing cells in a primate (non-human primate or human).In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of ICOS expressing cells in a primate (non-humanprimate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing cells in aprimate (non-human primate or human) more efficiently than that of theparental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventiondepletes ICOS expressing cells in a primate (non-human primate or human)more efficiently than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing cells in a primate (non-human primateor human) is at least about 2×, at least about 5×, at least about 10×,at least about 20×, at least about 50×, or at least about 100× lowerthan that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing cells in a primate (non-human primate orhuman) than that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention achieves at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 75%, at least about 100% higher depletion of ICOSexpressing cells in a primate (non-human primate or human) than that ofthe fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing cells in a primate (non-human primate orhuman) than that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention achieves at least about 2×, at least about 5×, at leastabout 10×, at least about 15×, at least about 20×, at least about 25×,at least about 50×, or at least about 100× higher depletion of ICOSexpressing cells in a primate (non-human primate or human) than that ofthe fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing T cells in a primate (non-human primate orhuman). In one embodiment, administration of one or more therapeuticdoses of an anti-ICOS antibody of the invention may achieve at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, at least about 99%,or at least about 100% depletion of ICOS expressing T cells in a primate(non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing T cells ina primate (non-human primate or human) more efficiently than that of theparental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventiondepletes ICOS expressing T cells in a primate (non-human primate orhuman) more efficiently than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing T cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing T cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing T cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing T cells in a primate (non-human primateor human) is at least about 2×, at least about 5×, at least about 10×,at least about 20×, at least about 50×, or at least about 100× lowerthan that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing T cells in a primate (non-human primate orhuman) than that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention achieves at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 75%, at least about 100% higher depletion of ICOSexpressing T cells in a primate (non-human primate or human) than thatof the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing T cells in a primate (non-human primate orhuman) than that of the parental anti-ICOS antibody (e.g., an antibodycomprising the same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention achieves at least about 2×, at least about 5×, at leastabout 10×, at least about 15×, at least about 20×, at least about 25×,at least about 50×, or at least about 100× higher depletion of ICOSexpressing T cells in a primate (non-human primate or human) than thatof the fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing T helper cells in a primate (non-human primateor human). In one embodiment, administration of one or more therapeuticdoses of an anti-ICOS antibody of the invention may achieve at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, at least about 99%,or at least about 100% depletion of ICOS expressing T helper cells in aprimate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing T helpercells in a primate (non-human primate or human) more efficiently thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention depletes ICOS expressing T helper cells in a primate(non-human primate or human) more efficiently than that of thefucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing T helper cells in a primate(non-human primate or human) than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inone embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing T helper cells in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing T helper cells in aprimate (non-human primate or human) is at least about 2×, at leastabout 5×, at least about 10×, at least about 20×, at least about 50×, orat least about 100× lower than that of the parental anti-ICOS antibody(e.g., an antibody comprising the same variable domain amino acidsequence, but having 1) a fucosylated Fc domain or 2) an Fc domain aminoacid sequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing T helper cells in a primate (non-humanprimate or human) is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing T helper cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing T helper cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing T helper cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing T helper cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing Th1 cells in a primate (non-human primate orhuman). In one embodiment, administration of one or more therapeuticdoses of an anti-ICOS antibody of the invention may achieve at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, at least about 99%,or at least about 100% depletion of ICOS expressing Th1 cells in aprimate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing Th1 cellsin a primate (non-human primate or human) more efficiently than that ofthe parental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventiondepletes ICOS expressing Th1 cells in a primate (non-human primate orhuman) more efficiently than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th1 cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th1 cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing Th1 cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing Th1 cells in a primate (non-humanprimate or human) is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th1 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th1 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th1 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th1 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing Th2 cells in a primate (non-human primate orhuman). In one embodiment, administration of one or more therapeuticdoses of an anti-ICOS antibody of the invention may achieve at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, at least about 99%,or at least about 100% depletion of ICOS expressing Th2 cells in aprimate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing Th2 cellsin a primate (non-human primate or human) more efficiently than that ofthe parental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventiondepletes ICOS expressing Th2 cells in a primate (non-human primate orhuman) more efficiently than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th2 cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th2 cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing Th2 cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing Th2 cells in a primate (non-humanprimate or human) is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th2 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th2 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th2 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th2 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing Th17 cells in a primate (non-human primate orhuman). In one embodiment, administration of one or more therapeuticdoses of an anti-ICOS antibody of the invention may achieve at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, at least about 99%,or at least about 100% depletion of ICOS expressing Th17 cells in aprimate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing Th17 cellsin a primate (non-human primate or human) more efficiently than that ofthe parental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventiondepletes ICOS expressing Th17 cells in a primate (non-human primate orhuman) more efficiently than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th17 cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing Th17 cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing Th17 cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing Th17 cells in a primate (non-humanprimate or human) is at least about 2×, at least about 5×, at leastabout 10×, at least about 20×, at least about 50×, or at least about100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th17 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing Th17 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th17 cells in a primate (non-human primateor human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing Th17 cells in a primate (non-human primateor human) than that of the fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete ICOS expressing memory helper T cells in a primate (non-humanprimate or human). In one embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention may achieveat least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%, or at least about 100% depletion of ICOS expressing memoryhelper T cells in a primate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing memoryhelper T cells in a primate (non-human primate or human) moreefficiently than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes ICOS expressing memoryhelper T cells in a primate (non-human primate or human) moreefficiently than that of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inone embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of ICOS expressing memory helper T cells ina primate (non-human primate or human) is at least about 2×, at leastabout 5×, at least about 10×, at least about 20×, at least about 50×, orat least about 100× lower than that of the parental anti-ICOS antibody(e.g., an antibody comprising the same variable domain amino acidsequence, but having 1) a fucosylated Fc domain or 2) an Fc domain aminoacid sequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inanother embodiment, administration of one or more therapeutic doses ofan anti-ICOS antibody of the invention achieves at least about 5%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 75%, at least about 100% higherdepletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inanother embodiment, administration of one or more therapeutic doses ofan anti-ICOS antibody of the invention achieves at least about 2×, atleast about 5×, at least about 10×, at least about 15×, at least about20×, at least about 25×, at least about 50×, or at least about 100×higher depletion of ICOS expressing memory helper T cells in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

Depletion of a particular cell type may lead to the depletion of asecreted product of said cell type. For example, depletion of Th17 cellsusing an effector function enhanced anti-ICOS antibody of the inventionmay lead to depletion of IL-17. The present invention also provides forantibodies that efficiently deplete IL-17 in a primate (non-humanprimate or human). In one embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention may achieveat least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, at leastabout 99%, or at least about 100% depletion of IL-17 in a primate(non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes IL-17 in a primate(non-human primate or human) more efficiently than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention depletesIL-17 in a primate (non-human primate or human) more efficiently thanthat of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of IL-17 in a primate (non-human primate or human) thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In one embodiment, administrationof one or more therapeutic doses of an anti-ICOS antibody of theinvention achieves at least about 2-fold, at least about 3-fold, atleast about 5-fold, or at least about 10-fold higher depletion of IL-17in a primate (non-human primate or human) than that of the fucosylatedJMab-136 anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of IL-17 in a primate (non-human primate orhuman) is at least about 2×, at least about 5×, at least about 10×, atleast about 20×, at least about 50×, or at least about 100× lower thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment, the EC50value of an anti-ICOS antibody of the invention for the depletion ofIL-17 in a primate (non-human primate or human) is at least about 2×, atleast about 5×, at least about 10×, at least about 20×, at least about50×, or at least about 100× lower than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of IL-17 in a primate (non-human primate or human) than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about75%, at least about 100% higher depletion of IL-17 in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of IL-17 in a primate (non-human primate or human) than thatof the parental anti-ICOS antibody (e.g., an antibody comprising thesame variable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 2×, at least about 5×, at least about 10×, atleast about 15×, at least about 20×, at least about 25×, at least about50×, or at least about 100× higher depletion of IL-17 in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete IL-2 in a primate (non-human primate or human). In oneembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of IL-2 in a primate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes IL-2 in a primate(non-human primate or human) more efficiently than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention depletesIL-2 in a primate (non-human primate or human) more efficiently thanthat of the fucosylated JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of IL-2 in a primate (non-human primate or human) thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In one embodiment, administrationof one or more therapeutic doses of an anti-ICOS antibody of theinvention achieves at least about 2-fold, at least about 3-fold, atleast about 5-fold, or at least about 10-fold higher depletion of IL-2in a primate (non-human primate or human) than that of the fucosylatedJMab-136 anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of IL-2 in a primate (non-human primate orhuman) is at least about 2×, at least about 5×, at least about 10×, atleast about 20×, at least about 50×, or at least about 100× lower thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment, the EC50value of an anti-ICOS antibody of the invention for the depletion ofIL-2 in a primate (non-human primate or human) is at least about 2×, atleast about 5×, at least about 10×, at least about 20×, at least about50×, or at least about 100× lower than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of IL-2 in a primate (non-human primate or human) than that ofthe parental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about75%, at least about 100% higher depletion of IL-2 in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of IL-2 in a primate (non-human primate or human) than that ofthe parental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In another embodiment, administration of oneor more therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 2×, at least about 5×, at least about 10×, atleast about 15×, at least about 20×, at least about 25×, at least about50×, or at least about 100× higher depletion of IL-2 in a primate(non-human primate or human) than that of the fucosylated JMab-136anti-ICOS antibody.

ICOS expressing T cells have been implicated in germinal centerformation in mouse model systems. Data disclosed herein demonstratesthat ICOS expressing cells are also involved in maintaining of thestructural integrity and B cell compartment of already formed germinalcenters. Without being bound by a particular model, the depletion ofICOS expressing T cells in a primate (non-human primate or human) byadministering one or more therapeutic doses of an anti-ICOS antibody ofthe invention may prevent the formation of germinal centers, may disruptthe architecture of already formed germinal centers, may depletegerminal center B cells from secondary lymphoid organs and/or maydeplete circulating class switched B cells. Germinal center formationmay be monitored by any method known in the art, for example, but notlimited to, histological examination of secondary lymphoid organs oranalysis of the lymphoid cells isolated from secondary lymphoid tissuesby flow cytometry. The disruption of germinal center architecture may bemonitored by any method known in the art, for example, but not limitedto, histological examination of secondary lymphoid organs. Depletion ofgerminal center B cells from secondary lymphoid organs may be monitoredby any method known in the art, for example, but not limited to,histological examination of secondary lymphoid organs or analysis of thelymphoid cells isolated from secondary lymphoid tissues by flowcytometry. Depletion of circulating class switched B cells may bemonitored by any method known in the art, for example, but not limitedto, analysis of circulating lymphoid cells by flow cytometry. Classswitched B cells may be identified based on their specific expression,or lack thereof, of cell surface markers, for example, but not limitedto, circulating class switched B cells may be identified asCD27+IgM-IgD-B cells.

The present invention provides for antibodies that efficiently preventgerminal center formation in a secondary lymphoid organ of a primate(non-human primate or human).

In one embodiment, the secondary lymphoid organ is a lymph node. Inanother embodiment, the secondary lymphoid organ is the spleen. In afurther embodiment, the secondary lymphoid organ is the tonsil. In oneembodiment, the secondary lymphoid organ is a mesenteric lymph node.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention prevents germinal centerformation in a secondary lymphoid organ of a primate (non-human primateor human) for at least 1 day, at least 2 days at least 5 days, at least1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 9 months. In a specific embodiment, thesecondary lymphoid organ is the spleen. In another specific embodiment,the secondary lymphoid organ is the tonsil.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention prevents germinal centerformation in a secondary lymphoid organ of a primate (non-human primateor human) for a longer time period than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inanother embodiment, administration of one or more therapeutic doses ofan anti-ICOS antibody of the invention prevents germinal centerformation in a secondary lymphoid organ of a primate (non-human primateor human) for a longer time period than that of the fucosylated JMAb-136anti-ICOS antibody. In a specific embodiment, the secondary lymphoidorgan is the spleen. In another specific embodiment, the secondarylymphoid organ is the tonsil.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention prevents germinal center formationin a secondary lymphoid organ of a primate (non-human primate or human)more efficiently than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention prevents germinal center formationin a secondary lymphoid organ of a primate (non-human primate or human)more efficiently than that of the fucosylated JMAb-136 anti-ICOSantibody.

The present invention also provides for antibodies that efficientlydisrupt germinal center architecture in a secondary lymphoid organ of aprimate (non-human primate or human). In one embodiment, the secondarylymphoid organ is a lymph node. In another embodiment, the secondarylymphoid organ is the spleen. In a further embodiment, the secondarylymphoid organ is the tonsil. In one embodiment, the secondary lymphoidorgan is a mesenteric lymph node.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention disrupts germinal centerarchitecture in a secondary lymphoid organ of a primate (non-humanprimate or human) for at least 1 day, at least 2 days at least 5 days,at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month,at least 2 months, at least 3 months, at least 4 months, at least 5months, at least 6 months, at least 9 months. In a specific embodiment,the secondary lymphoid organ is the spleen. In another specificembodiment, the secondary lymphoid organ is the tonsil.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention disrupts germinal centerarchitecture in a secondary lymphoid organ of a primate (non-humanprimate or human) for a longer time period than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention disruptsgerminal center architecture in a secondary lymphoid organ of a primate(non-human primate or human) for a longer time period than that of thefucosylated JMAb-136 anti-ICOS antibody. In a specific embodiment, thesecondary lymphoid organ is the spleen. In another specific embodiment,the secondary lymphoid organ is the tonsil.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention disrupts germinal centerarchitecture in a secondary lymphoid organ of a primate (non-humanprimate or human) more efficiently than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inanother embodiment, administration of one or more therapeutic doses ofan anti-ICOS antibody of the invention disrupts germinal centerarchitecture in a secondary lymphoid organ of a primate (non-humanprimate or human) more efficiently than that of the fucosylated JMAb-136anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete germinal center B cells from a secondary lymphoid organ in aprimate (non-human primate or human). In one embodiment, the secondarylymphoid organ is a lymph node. In another embodiment, the secondarylymphoid organ is the spleen. In a further embodiment, the secondarylymphoid organ is the tonsil. In one embodiment, the secondary lymphoidorgan is a mesenteric lymph node.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention depletes germinal center Bcells from a secondary lymphoid organ in a primate (non-human primate orhuman) for at least 1 day, at least 2 days at least 5 days, at least 1week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 9 months. In a specific embodiment, thesecondary lymphoid organ is the spleen. In another specific embodiment,the secondary lymphoid organ is the tonsil. Depletion of germinal centerB cells is considered to “substantially persist” during the time periodfollowing the administration of one or more doses of anti-ICOS antibodywhen the number of germinal center B cells is at least 10% lower in theantibody treated sample than the number of germinal center B cells inthe untreated control sample.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention depletes germinal center Bcells from a secondary lymphoid organ in a primate (non-human primate orhuman) for a longer time period than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inanother embodiment, administration of one or more therapeutic doses ofan anti-ICOS antibody of the invention depletes germinal center B cellsfrom a secondary lymphoid organ in a primate (non-human primate orhuman) for a longer time period than that of the fucosylated JMAb-136anti-ICOS antibody. In a specific embodiment, the secondary lymphoidorgan is the spleen. In another specific embodiment, the secondarylymphoid organ is the tonsil.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of germinal center B cells from a secondarylymphoid organ in a primate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes germinal center B cellsfrom a secondary lymphoid organ in a primate (non-human primate orhuman) more efficiently than that of the parental anti-ICOS antibody(e.g., an antibody comprising the same variable domain amino acidsequence, but having 1) a fucosylated Fc domain or 2) an Fc domain aminoacid sequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes germinal center B cellsfrom a secondary lymphoid organ in a primate (non-human primate orhuman) more efficiently than that of the fucosylated JMAb-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of germinal center B cells from a secondary lymphoidorgan in a primate (non-human primate or human) than that of theparental anti-ICOS antibody (e.g., an antibody comprising the samevariable domain amino acid sequence, but having 1) a fucosylated Fcdomain or 2) an Fc domain amino acid sequence, which has not beenmodified to increase ADCC). In one embodiment, administration of one ormore therapeutic doses of an anti-ICOS antibody of the inventionachieves at least about 2-fold, at least about 3-fold, at least about5-fold, or at least about 10-fold higher depletion of germinal center Bcells from a secondary lymphoid organ in a primate (non-human primate orhuman) than that of the fucosylated the JMab-136 anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of germinal center B cells from a secondarylymphoid organ in a primate (non-human primate or human) is at leastabout 2×, at least about 5×, at least about 10×, at least about 20×, atleast about 50×, or at least about 100× lower than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, the EC50 value of an anti-ICOSantibody of the invention for the depletion of germinal center B cellsfrom a secondary lymphoid organ in a primate (non-human primate orhuman) is at least about 2×, at least about 5×, at least about 10×, atleast about 20×, at least about 50×, or at least about 100× lower thanthat of the parental anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of germinal center B cells from a secondary lymphoid organ ina primate (non-human primate or human) than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention achieves atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 75%, atleast about 100% higher depletion of germinal center B cells from asecondary lymphoid organ in a primate (non-human primate or human) thanthat of the fucosylated the JMab-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of germinal center B cells from a secondary lymphoid organ ina primate (non-human primate or human) than that of the parentalanti-ICOS antibody (e.g., an antibody comprising the same variabledomain amino acid sequence, but having 1) a fucosylated Fc domain or 2)an Fc domain amino acid sequence, which has not been modified toincrease ADCC). In another embodiment, administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention achieves atleast about 2×, at least about 5×, at least about 10×, at least about15×, at least about 20×, at least about 25×, at least about 50×, or atleast about 100× higher depletion of germinal center B cells from asecondary lymphoid organ in a primate (non-human primate or human) thanthat of the fucosylated JMab-136 anti-ICOS antibody.

The present invention also provides for antibodies that efficientlydeplete circulating class switched B cells in a primate (non-humanprimate or human). In one embodiment, the administration of one or moretherapeutic doses of an anti-ICOS antibody of the invention depletescirculating class switched B cells in a primate (non-human primate orhuman) for at least 1 day, at least 2 days at least 5 days, at least 1week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 9 months. Depletion of circulating classswitched B cells is considered to “substantially persist” during thetime period following the administartion of one or more doses ofanti-ICOS antibody when the number of circulating class switched B cellsis at least 10% lower in the antibody treated sample than the number ofcirculating class switched B cells in the untreated control sample.

In one embodiment, the administration of one or more therapeutic dosesof an anti-ICOS antibody of the invention depletes circulating classswitched B cells in a primate (non-human primate or human) for a longertime period than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes circulating class switchedB cells in a primate (non-human primate or human) for a longer timeperiod than that of the fucosylated JMAb-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may achieve at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 99%, or at leastabout 100% depletion of circulating class switched B cells in a primate(non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention may deplete circulating classswitched B cells to less than 2%, less than 1.5%, less than 1%, lessthan 0.9%, less than 0.8%, less than 07%, less than 0.6%, less than0.5%, less than 0.4%, less than 0.3% or less than 0.1% of peripheralblood lymphocytes (PBL) in a primate (non-human primate or human).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention depletes circulating class switchedB cells in a primate (non-human primate or human) more efficiently thanthat of the parental anti-ICOS antibody (e.g., an antibody comprisingthe same variable domain amino acid sequence, but having 1) afucosylated Fc domain or 2) an Fc domain amino acid sequence, which hasnot been modified to increase ADCC). In another embodiment,administration of one or more therapeutic doses of an anti-ICOS antibodyof the invention depletes circulating class switched B cells in aprimate (non-human primate or human) more efficiently than that of thefucosylated JMAb-136 anti-ICOS antibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of circulating class switched B cells in a primate(non-human primate or human) than that of the parental anti-ICOSantibody (e.g., an antibody comprising the same variable domain aminoacid sequence, but having 1) a fucosylated Fc domain or 2) an Fc domainamino acid sequence, which has not been modified to increase ADCC). Inone embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2-fold, atleast about 3-fold, at least about 5-fold, or at least about 10-foldhigher depletion of circulating class switched B cells in a primate(non-human primate or human) than that of the fucosylated the JMab-136anti-ICOS antibody.

In one embodiment, the EC50 value of an anti-ICOS antibody of theinvention for the depletion of circulating class switched B cells in aprimate (non-human primate or human) is at least about 2×, at leastabout 5×, at least about 10×, at least about 20×, at least about 50×, orat least about 100× lower than that of the parental anti-ICOS antibody(e.g., an antibody comprising the same variable domain amino acidsequence, but having 1) a fucosylated Fc domain or 2) an Fc domain aminoacid sequence, which has not been modified to increase ADCC). In anotherembodiment, the EC50 value of an anti-ICOS antibody of the invention forthe depletion of circulating class switched B cells in a primate(non-human primate or human) is at least about 2×, at least about 5×, atleast about 10×, at least about 20×, at least about 50×, or at leastabout 100× lower than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC).

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of circulating class switched B cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 5%, at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 100% higherdepletion of circulating class switched B cells in a primate (non-humanprimate or human) than that of the fucosylated the JMab-136 anti-ICOSantibody.

In one embodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of circulating class switched B cells in a primate (non-humanprimate or human) than that of the parental anti-ICOS antibody (e.g., anantibody comprising the same variable domain amino acid sequence, buthaving 1) a fucosylated Fc domain or 2) an Fc domain amino acidsequence, which has not been modified to increase ADCC). In anotherembodiment, administration of one or more therapeutic doses of ananti-ICOS antibody of the invention achieves at least about 2×, at leastabout 5×, at least about 10×, at least about 15×, at least about 20×, atleast about 25×, at least about 50×, or at least about 100× higherdepletion of circulating class switched B cells in a primate (non-humanprimate or human) than that of the fucosylated JMab-136 anti-ICOSantibody.

In one embodiment, an anti-ICOS antibody described herein mediatesantibody-dependent cellular cytotoxicity (ADCC), complement-dependentcell-mediated cytotoxicity (CDC), and/or antibody-dependentphagocytosis. In one embodiment, an anti-ICOS antibody of the inventionmediates antibody-dependent cellular cytotoxicity (ADCC) and/orantibody-dependent phagocytosis. In one embodiment, an anti-ICOSantibody of the invention has enhanced antibody-dependent cellularcytotoxicity (ADCC).

In one embodiment, an anti-ICOS antibody of the invention comprises avariant Fc region that mediates enhanced antibody-dependent cellularcytotoxicity (ADCC). In a further embodiment, an anti-ICOS antibody ofthe invention comprises a variant Fc region comprising at least onesubstitution of an amino acid residue selected from the group consistingof: residue 239, 330, and 332, wherein the amino acid residue positionsare determined according to the EU convention. In a specific embodiment,an anti-ICOS antibody of the invention comprises a variant Fc regioncomprising at least on amino acid substitution selected from the groupconsisting of: S239D, A330L, and 1332E; wherein the amino acid residuepositions are determined according to the EU convention. In a furtherembodiment, an anti-ICOS antibody of the invention comprises at leastone amino acid residue selected from the group consisting of: D atposition 239, L at position 330, and E at position 332; wherein theamino acid residue positions are determined according to the EUconvention.

In one embodiment, an anti-ICOS antibody of the invention comprises anengineered Fc region comprising at least one engineered glycoform,wherein said engineered Fc region mediates enhanced antibody-dependentcellular cytotoxicity (ADCC). In one embodiment, an anti-ICOS antibodyof the inventions comprises an engineered Fc region lackingglycosylation. In one embodiment, an anti-ICOS antibody of the inventioncomprises an engineered Fc region having complex N-glycoside-linkedsugar chains linked to Asn297 in which fucose is not bound toN-acetylglucosamine in the reducing end.

In certain embodiments, an anti-ICOS antibody of the invention comprisesa variant Fc region that has a higher affinity for an Fc binding proteinsuch as, but not limited to, Fc receptor, C1q than a wild type Fcregion. In one embodiment, an anti-ICOS antibody of the inventioncomprises a variant Fc region that has higher affinity for the FcγRIIIAreceptor protein than a wild type Fc region.

In certain embodiments, an anti-ICOS antibody of the invention comprisesan engineered Fc region comprising at least one engineered glycoform,wherein said engineered Fc region has a higher affinity for an Fcbinding protein such as, but not limited to, Fc receptor, C1q than awild type Fc region. In one embodiment, an anti-ICOS antibody of theinvention comprises an engineered Fc region comprising at least oneengineered glycoform, wherein said engineered Fc region has higheraffinity for the FcγRIIIA receptor protein than a wild type Fc region.

The present invention also relates to methods of treating and preventingT cell-mediated diseases and disorders, such as, but not limited to,chronic infection, autoimmune disease or disorder, inflammatory diseaseor disorder, graft-versus-host disease (GVHD), transplant rejection, andT cell proliferative disorder in a human, comprising administering to ahuman in need thereof an anti-ICOS antibody with enhanced effectorfunction (e.g., antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cell-mediated cytotoxicity (CDC), and/orantibody-dependent phagocytosis) in an amount sufficient to depletecirculating ICOS expressing cells. In a particular aspect, the presentinvention also concerns methods of treating and preventing Tcell-mediated diseases and disorders, such as, but not limited to,chronic infection, autoimmune disease or disorder, inflammatory diseaseor disorder, graft-versus-host disease (GVHD), transplant rejection, andT cell proliferative disorder in a human comprising administration of atherapeutically effective regimen of an anti-ICOS antibody with enhancedeffector function, which is of the IgG1 or IgG3 human isotype.

The invention encompasses methods of identifying, diagnosing, treating,and monitoring disease progression in patients. The patient may have thedisease, disorder, or condition as a result of experimental research,e.g., it may be an experimental model developed for the disease,disorder, or condition. Alternatively, the patient may have the disease,disorder, or condition in the absence of experimental manipulation.Patients include humans, mice, rats, horses, pigs, cats, dogs, and anyanimal used for research.

The patient may comprise a differentially regulated ICOS mRNA or ICOSLmRNA or miR-101 level. A differentially regulated ICOS mRNA or ICOSLmRNA or miR-101 level may be one in which a tissue sample of the patientexhibits increased expression of ICOS mRNA or ICOSL mRNA or miR-101relative to a control tissue sample of the patient or relative to ahealthy control individual. A differentially regulated ICOS mRNA orICOSL mRNA or miR-101 level may be one in which a tissue sample of thepatient exhibits decreased expression of ICOS mRNA or ICOSL mRNA ormiR-101 relative to a control sample of the patient or relative to ahealthy control individual. The differential increase or decrease inexpression may be approximately 10%-500% of the control sample,approximately 10%-400% of the control sample, approximately 10%-300% ofthe control sample, approximately 10%-250% of the control sample,approximately 10%-200% of the control sample, approximately 10%-150% ofthe control sample, approximately 10%-100% of the control sample,approximately 10%-50% of the control sample, approximately 100%-500% ofthe control sample, approximately 200%-500% of the control sample,approximately 300%-500% of the control sample, approximately 400%-500%of the control sample, approximately 50%-100% of the control sample,approximately 100%-200% of the control sample, approximately 100%-400%of the control sample, approximately 200%-400% of the control sample,approximately 10%-50% of the control sample, approximately 20%-100% ofthe control sample, approximately 25%-75% of the control sample, orapproximately 50%-100% of the control sample. It may be 10, 20, 25, 30,40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500 percent ofthe control sample.

Administration of an anti-ICOS antibody of the invention may result inneutralization of the differentially regulated ICOS mRNA or ICOSL mRNAor miR-101 level. Neutralization of the differentially regulated ICOSmRNA or ICOSL mRNA or miR-101 level may be a reduction of at least 2%,at least 3%, at least 4%, at least 5%, at least 7%, at least 8%, atleast 10%, at least 15%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, or at least 90% of ICOS mRNA or ICOSL mRNA ormiR-101 level. Alternatively, neutralization of the differentiallyregulated ICOS mRNA or ICOSL mRNA or miR-101 level refers to a reductionof expression of up-regulated ICOS mRNA or ICOSL mRNA or miR-101 that iswithin at most 50%, at most 45%, at most 40%, at most 35%, at most 30%,at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, at most4%, at most 3%, at most 2%, or at most 1% of expression levels of theICOS mRNA or ICOSL mRNA or miR-101 level in a control sample.

The upregulation or downregulation of the ICOS mRNA or ICOSL mRNA ormiR-101 in the patient may be by any degree relative to that of a samplefrom a control (which may be from a sample that is not disease tissue ofthe patient (e.g., non-lesional skin of a SLE patient) or from a healthyperson not afflicted with the disease or disorder). The degreeupregulation or downregulation may be at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 90%, atleast 100%, at least 125%, at least 150%, or at least 200%, or at least300%, or at least 400%, or at least 500% that of the control or controlsample.

In methods of monitoring or prognosing disease progression of a patient,samples from the patient may be obtained before and after administrationof an agent.

Samples include any biological fluid or tissue, such as whole blood,serum, muscle, saliva, urine, synovial fluid, bone marrow, cerebrospinalfluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavagefluid, peripheral blood mononuclear cells, total white blood cells,lymph node cells, spleen cells, tonsil cells, or skin. The samples maybe obtained by any means known in the art.

ICOS mRNA or ICOSL mRNA or miR-101 levels are obtained in the (beforeand after agent administration) samples. The ICOS mRNA or ICOSL mRNA ormiR-101 levels in the samples are compared.

The sample obtained from the patient may be obtained prior to a firstadministration of the agent, i.e., the patient is naïve to the agent.Alternatively, the sample obtained from the patient may occur afteradministration of the agent in the course of treatment. For example, theagent may have been administered prior to the initiation of themonitoring protocol. Following administration of the agent an additionalsamples may be obtained from the patient. The samples may be of the sameor different type, e.g., each sample obtained may be a blood sample, oreach sample obtained may be a serum sample. The ICOS mRNA or ICOSL mRNAor miR-101 levels detected in each sample may be the same, may overlapsubstantially, or may be similar.

The samples may be obtained at any time before and after theadministration of the therapeutic agent. The sample obtained afteradministration of the therapeutic agent may be obtained at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 12, or at least 14 days afteradministration of the therapeutic agent. The sample obtained afteradministration of the therapeutic agent may be obtained at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, or at least 8weeks after administration of the therapeutic agent. The sample obtainedafter administration of the therapeutic agent may be obtained at least2, at least 3, at least 4, at least 5, or at least 6 months followingadministration of the therapeutic agent.

Additional samples may be obtained from the patient followingadministration of the therapeutic agent. At least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 12, at least 15, at least 20, at least 25 samples maybe obtained from the patient to monitor progression or regression of thedisease or disorder over time. Disease progression may be monitored overa time period of at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 1 year, at least 2 years, at least 3 years,at least 4 years, at least 5 years, at least 10 years, or over thelifetime of the patient. Additional samples may be obtained from thepatient at regular intervals such as at monthly, bimonthly, once aquarter year, twice a year, or yearly intervals. The samples may beobtained from the patient following administration of the agent atregular intervals. For instance, the samples may be obtained from thepatient at one week following each administration of the agent, or attwo weeks following each administration of the agent, or at three weeksfollowing each administration of the agent, or at one month followingeach administration of the agent, or at two months following eachadministration of the agent. Alternatively, multiple samples may beobtained from the patient following each administration of the agent.

The invention also encompasses methods employing ICOS mRNA or ICOSL mRNAor miR-101 levels to treat, diagnose, prognose, and monitor myositis.The ICOS mRNA or ICOSL mRNA or miR-101 levels can also be used to guidedosage and treatment of myositis patients or models of myositis disease.

5.1. Monoclonal Anti-ICOS Antibodies

A monoclonal anti-ICOS antibody exhibits binding specificity to humanICOS antigen and may mediate human ADCC, CDC and/or antibody-dependentphagocytosis. Such an antibody can be generated using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. Antibodies arehighly specific, being directed against a single antigenic site. Anengineered anti-ICOS antibody can be produced by any means known in theart, including, but not limited to, those techniques described below andimprovements to those techniques. Large-scale high-yield productiontypically involves culturing a host cell that produces the engineeredanti-ICOS antibody and recovering the anti-ICOS antibody from the hostcell culture.

5.1.1. Hybridoma Technique

Monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling et al., in Monoclonal Antibodies and TCell Hybridomas, 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated herein by reference in their entireties). For example, inthe hybridoma method, a mouse or other appropriate host animal, such asa hamster or macaque monkey, is immunized to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Lymphocytes may also beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from MOPC-21 and MPC-11 mouse tumors available from theSalk Institute Cell Distribution Center, San Diego, Calif., USA, andSP-2 or X63-Ag8.653 cells available from the American Type CultureCollection, Rockville, Md., USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the human ICOSantigen. The binding specificity of monoclonal antibodies produced byhybridoma cells can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI 1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

5.1.2. Recombinant DNA Techniques

DNA encoding an anti-ICOS antibody described herein is readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of anti-ICOS antibodies). Thehybridoma cells serve as a source of such DNA. Once isolated, the DNAmay be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of anti-ICOS antibodiesin the recombinant host cells.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of affected tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector. The vector is electroporated in E. coli and the E. coliis infected with helper phage. Phage used in these methods is typicallyfilamentous phage including fd and M13 and the V_(H) and V_(L) domainsare usually recombinantly fused to either the phage gene III or geneVIII. Phage expressing an antigen-binding domain that binds to aparticular antigen can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J.Immunol. Methods, 184:177-186; Kettleborough et al, 1994, Eur. J.Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton etal., 1994, Advances in Immunology, 57:191-280; International ApplicationNo. PCT/GB91/O1 134; International Publication Nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen-binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)₂ fragments can also be employed using methods knownin the art such as those disclosed in PCT Publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995,AJRI, 34:26-34; and Better et al., 1988, Science, 240:1041-1043 (saidreferences incorporated by reference in their entireties).

Antibodies may be isolated from antibody phage libraries generated usingthe techniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991). Marks et al., J.Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Chain shufflingcan be used in the production of high affinity (nM range) humanantibodies (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al, Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of anti-ICOS antibodies.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. The vectors for expressing the VH or VL domains may comprise anEF-1α promoter, a secretion signal, a cloning site for the variabledomain, constant domains, and a selection marker such as neomycin. TheVH and VL domains may also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

5.2. Chimeric Antibodies

The anti-ICOS antibodies herein specifically include chimeric antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while another portion of the chain(s) is identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a nonhuman primate (e.g.,Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and humanconstant region sequences (U.S. Pat. No. 5,693,780).

5.3.Altered/Mutant Antibodies

Anti-ICOS antibodies of compositions and methods described herein can bemutant antibodies. As used herein, “antibody mutant” or “alteredantibody” refers to an amino acid sequence variant of an anti-ICOSantibody wherein one or more of the amino acid residues of an anti-ICOSantibody have been modified. The modifications to the amino acidsequence of an anti-ICOS antibody include modifications to the sequencethat may improve affinity or avidity of the antibody for its antigen,and/or modifications to the Fc portion of the antibody that may improveeffector function.

The present invention therefore relates to anti-ICOS antibodies withenhanced effector function disclosed herein as well as altered/mutantderivatives thereof including, but not limited to ones exhibitingaltered human ICOS binding characteristics; e.g. altered associationconstants k_(ON), dissociation constants k_(OFF), and/or equilibriumconstant or binding affinity, K_(D). In certain embodiments the K_(D) ofan anti-ICOS antibody described herein, or an altered/mutant derivativethereof, for human ICOS may be no more than about 10⁻⁶M, 10⁻⁷M, 10⁻⁸M,or 10⁻⁹M. Methods and reagents suitable for determination of suchbinding characteristics of an antibody of the present invention, or analtered/mutant derivative thereof, are known in the art and/or arecommercially available (se above and, e.g., U.S. Pat. No. 6,849,425,U.S. Pat. No. 6,632,926, U.S. Pat. No. 6,294,391, and U.S. Pat. No.6,143,574, each of which is hereby incorporated by reference in itsentirety). Moreover, equipment and software designed for such kineticanalyses are commercially available (e.g. Biacore® A100, and Biacore®2000 instruments; Biacore International AB, Uppsala, Sweden).

The modifications may be made to any known anti-ICOS antibodies oranti-ICOS antibodies identified as described herein. Such alteredantibodies necessarily have less than 100% sequence identity orsimilarity with a known anti-ICOS antibody. By way of example, analtered antibody may have an amino acid sequence that is within therange of from about 25% to about 95% identical or similar to the aminoacid sequence of either the heavy or light chain variable domain of ananti-ICOS antibody as described herein. An altered antibody may have anamino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%,85%, 90%, or 95% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof an anti-ICOS antibody as described herein. In another embodiment, analtered antibody may have an amino acid sequence having at least 25%,35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequenceidentity or similarity with the amino acid sequence of the heavy chainCDR1, CDR2, or CDR3 of an anti-ICOS antibody as described herein. In oneembodiment, an altered antibody may maintain human ICOS bindingcapability. In certain embodiments, an anti-ICOS antibody as describedherein may comprise a VH that is at least or about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore identical to the amino acid sequence of SEQ ID NO:7.

In another embodiment, an altered antibody may have an amino acidsequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or95% amino acid sequence identity or similarity with the amino acidsequence of the light chain CDR1, CDR2, or CDR3 of an anti-ICOS antibodyas described herein. In certain embodiments, an anti-ICOS antibody ofthe invention may comprise a VL that is at least or about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or more identical to an amino acid sequence of SEQ ID NO:2.

Identity or similarity with respect to a sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) or similar (i.e., amino acid residue fromthe same group based on common side-chain properties, see below) withanti-ICOS antibody residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence outside of thevariable domain shall be construed as affecting sequence identity orsimilarity.

“% identity,” as known in the art, is a measure of the relationshipbetween two polynucleotides or two polypeptides, as determined bycomparing their sequences. In general, the two sequences to be comparedare aligned to give a maximum correlation between the sequences. Thealignment of the two sequences is examined and the number of positionsgiving an exact amino acid or nucleotide correspondence between the twosequences determined, divided by the total length of the alignment andmultiplied by 100 to give a % identity figure. This % identity figuremay be determined over the whole length of the sequences to be compared,which is particularly suitable for sequences of the same or very similarlength and which are highly homologous, or over shorter defined lengths,which is more suitable for sequences of unequal length or which have alower level of homology.

For example, sequences can be aligned with the software clustalw underUnix which generates a file with an “.aln” extension, this file can thenbe imported into the Bioedit program (Hall, T. A. 1999, BioEdit: auser-friendly biological sequence alignment editor and analysis programfor Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98) which opens the.aln file. In the Bioedit window, one can choose individual sequences(two at a time) and alignment them. This method allows for comparison ofthe entire sequence.

Methods for comparing the identity of two or more sequences are wellknown in the art. Thus for instance, programs are available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux J. et al.,Nucleic Acids Res., 12:387-395, 1984, available from Genetics ComputerGroup, Madison, Wis., USA). The determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identitybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of Smith and Waterman (Advances in Applied Mathematics,2:482-489, 1981) and finds the best single region of similarity betweentwo sequences. BESTFIT is more suited to comparing two polynucleotide ortwo polypeptide sequences which are dissimilar in length, the programassuming that the shorter sequence represents a portion of the longer.In comparison, GAP aligns two sequences finding a “maximum similarity”according to the algorithm of Neddleman and Wunsch (J. Mol. Biol.,48:443-354, 1970). GAP is more suited to comparing sequences which areapproximately the same length and an alignment is expected over theentire length. Preferably the parameters “Gap Weight” and “LengthWeight” used in each program are 50 and 3 for polynucleotides and 12 and4 for polypeptides, respectively. Preferably % identities andsimilarities are determined when the two sequences being compared areoptimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Karlin & Altschul, 1990, Proc. Natl. Acad. Sci. USA,87:2264-2268, modified as in Karlin & Altschul, 1993, Proc. Natl. Acad.Sci. USA, 90:5873-5877, available from the National Center forBiotechnology Information (NCB), Bethesda, Md., USA and accessiblethrough the home page of the NCBI at www.ncbi.nlm.nih.gov). Theseprograms are non-limiting examples of a mathematical algorithm utilizedfor the comparison of two sequences. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.Biol., 215:403-410. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecule encoding all or a portion if ananti-ICOS antibody of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to a protein molecule of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.,25:3389-3402. PSI-Blast can also be used to perform an iterated searchwhich detects distant relationships between molecules (Id.). Whenutilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. See, http://www.ncbi.nlm.nih.gov.

Another non-limiting example of a program for determining identityand/or similarity between sequences known in the art is FASTA (PearsonW. R. and Lipman D. J., Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988,available as part of the Wisconsin Sequence Analysis Package).Preferably the BLOSUM62 amino acid substitution matrix (Henikoff S. andHenikoff J. G., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) isused in polypeptide sequence comparisons including where nucleotidesequences are first translated into amino acid sequences beforecomparison.

Yet another non-limiting example of a program known in the art fordetermining identity and/or similarity between amino acid sequences isSeqWeb Software (a web-based interface to the GCG Wisconsin Package: Gapprogram) which is utilized with the default algorithm and parametersettings of the program: blosum62, gap weight 8, length weight 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

Preferably the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to apolynucleotide or a polypeptide sequence of the present invention, thequery and the reference sequence being optimally aligned and theparameters of the program set at the default value.

To generate an altered antibody, one or more amino acid alterations(e.g., substitutions) are introduced in one or more of the hypervariableregions of the species-dependent antibody. One or more alterations(e.g., substitutions) of framework region residues may also beintroduced in an anti-ICOS antibody where these result in an improvementin the binding affinity of the antibody mutant for the antigen from thesecond mammalian species. Examples of framework region residues tomodify include those which non-covalently bind antigen directly (Amit etal., Science, 233:747-753 (1986)); interact with/effect the conformationof a CDR (Chothia et al., J. Mol. Biol., 196:901-917 (1987)); and/orparticipate in the VL-VH interface (EP 239 400B1). In certainembodiments, modification of one or more of such framework regionresidues results in an enhancement of the binding affinity of theantibody for the antigen from the second mammalian species. For example,from about one to about five framework residues may be altered in thisembodiment of the invention. Sometimes, this may be sufficient to yieldan antibody mutant suitable for use in preclinical trials, even wherenone of the hypervariable region residues have been altered. Normally,however, an altered antibody will comprise additional hypervariableregion alteration(s).

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of an anti-ICOSantibody for the antigen from the second mammalian species is such thatsuch randomly produced altered antibody can be readily screened.

One useful procedure for generating such an altered antibody is called“alanine scanning mutagenesis” (Cunningham and Wells, Science,244:1081-1085 (1989)). Here, one or more of the hypervariable regionresidue(s) are replaced by alanine or polyalanine residue(s) to affectthe interaction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing additional or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The Ala-mutants produced this way arescreened for their biological activity as described herein.

Another procedure for generating such an altered antibody involvesaffinity maturation using phage display (Hawkins et al., J. Mol. Biol.,254:889-896 (1992) and Lowman et al., Biochemistry, 30(45):10832-10837(1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites)are mutated to generate all possible amino acid substitutions at eachsite. The antibody mutants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedmutants are then screened for their biological activity (e.g., bindingaffinity) as herein disclosed.

Mutations in antibody sequences may include substitutions, deletions,including internal deletions, additions, including additions yieldingfusion proteins, or conservative substitutions of amino acid residueswithin and/or adjacent to the amino acid sequence, but that result in a“silent” change, in that the change produces a functionally equivalentanti-ICOS antibody. Conservative amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. In addition, glycine and proline are residues that caninfluence chain orientation. Non-conservative substitutions will entailexchanging a member of one of these classes for a member of anotherclass. Furthermore, if desired, non-classical amino acids or chemicalamino acid analogs can be introduced as a substitution or addition intothe antibody sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Any technique for mutagenesis known in the art can be used to modifyindividual nucleotides in a DNA sequence, for purposes of making aminoacid substitution(s) in the antibody sequence, or for creating/deletingrestriction sites to facilitate further manipulations. Such techniquesinclude, but are not limited to, chemical mutagenesis, in vitrosite-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488(1985); Hutchinson, C. et al., J. Biol. Chem., 253:6551 (1978)),oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet.,19:423-463 (1985); Hill et al., Methods Enzymol., 155:558-568 (1987)),PCR-based overlap extension (Ho et al., Gene, 77:51-59 (1989)),PCR-based megaprimer mutagenesis (Sarkar et al, Biotechniques, 8:404-407(1990)), etc. Modifications can be confirmed by double-stranded dideoxyDNA sequencing.

In certain embodiments of the invention, an anti-ICOS antibody can bemodified to produce fusion proteins; i.e., the antibody, or a fragmentthereof, fused to a heterologous protein, polypeptide or peptide. Incertain embodiments, the protein fused to the portion of an anti-ICOSantibody is an enzyme component of Antibody-Directed Enzyme ProdrugTherapy (ADEPT). Examples of other proteins or polypeptides that can beengineered as a fusion protein with an anti-ICOS antibody include, butare not limited to toxins such as ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed anti-viral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell, 47:641 (1986), and Goldenberg et al,Cancer Journal for Clinicians, 44:43 (1994). Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,non-binding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, WO 93/21232 published Oct. 28, 1993.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of the anti-ICOS antibody or fragmentsthereof (e.g., an antibody or a fragment thereof with higher affinitiesand lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997,Curr. Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson et al., 1999, J. Mol. Biol., 287:265-76; andLorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of thesepatents and publications are hereby incorporated by reference in itsentirety). The antibody can further be a binding-domain immunoglobulinfusion protein as described in U.S. Publication 20030118592, U.S.Publication 200330133939, and PCT Publication WO 02/056910, all toLedbetter et al., which are incorporated herein by reference in theirentireties.

5.4. Domain Antibodies

Anti-ICOS antibodies of compositions and methods of the invention can bedomain antibodies, e.g., antibodies containing the small functionalbinding units of antibodies, corresponding to the variable regions ofthe heavy (V_(H)) or light (V_(L)) chains of human antibodies. Examplesof domain antibodies include, but are not limited to, those availablefrom Domantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge,Mass., USA) that are specific to therapeutic targets (see, for example,WO04/058821; WO04/003019; U.S. Pat. Nos. 6,291,158; 6,582,915;6,696,245; and 6,593,081). Commercially available libraries of domainantibodies can be used to identify anti-ICOS domain antibodies. Incertain embodiments, anti-ICOS antibodies comprise an ICOS functionalbinding unit and a Fc gamma receptor functional binding unit.

In one embodiment, an anti-ICOS domain antibody may comprise any one of,or any combination of the CDRs of the heavy or light chains of theJMab-136 monoclonal antibody.

In another embodiment, an anti-ICOS domain antibody may comprise VH CDR3of JMab-136 together with any combination of the CDRs comprised by theheavy or light chains variable regions of the JMab-136 monoclonalantibody. An anti-ICOS domain antibody may also comprise VK CDR3 ofJMab-136 together with any combination of the CDRs comprised by theheavy or light chains variable regions of the JMab-136 monoclonalantibody.

In yet another embodiment, an anti-ICOS domain antibody may comprise VHCDR3 of JMab-136. An anti-ICOS domain antibody may also comprise VK CDR3of JMab-136.

5.5. Diabodies

In certain embodiments of the invention, anti-ICOS antibodies are“diabodies”. The term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

5.6. Vaccibodies

In certain embodiments of the invention, anti-ICOS antibodies areVaccibodies. Vaccibodies are dimeric polypeptides. Each monomer of avaccibody consists of a scFv with specificity for a surface molecule onAPC connected through a hinge region and a Cγ3 domain to a second scFv.In other embodiments of the invention, vaccibodies containing as one ofthe scFv's an anti-ICOS antibody fragment may be used to juxtapose thoseICOS expressing cells to be destroyed and an effector cell that mediatesADCC. For example, see, Bogen et al., U.S. Patent ApplicationPublication No. 20040253238.

5.7. Linear Antibodies

In certain embodiments of the invention, anti-ICOS antibodies are linearantibodies. Linear antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen-bindingregions. Linear antibodies can be bispecific or monospecific. See,Zapata et al., Protein Eng., 8(10):1057-1062 (1995).

5.8. Parent Antibody

In certain embodiments of the invention, an anti-ICOS antibody is aparent antibody. A “parent antibody” is an antibody comprising an aminoacid sequence which may lack, or may be deficient in, one or more aminoacid residues in or adjacent to one or more hypervariable regionsthereof compared to an altered/mutant antibody as herein disclosed.Thus, the parent antibody may have a shorter hypervariable region thanthe corresponding hypervariable region of an antibody mutant as hereindisclosed. The parent polypeptide may comprise a native antibodysequence (i.e., a naturally occurring, including a naturally occurringallelic variant) or an antibody sequence with pre-existing amino acidsequence modifications (such as other insertions, deletions and/orsubstitutions) of a naturally occurring sequence. The parent antibodymay be a humanized antibody or a human antibody.

5.9. Antibody Fragments

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods, 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above. Fab′-SHfragments can also be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology,10:163-167 (1992)). According to another approach, F(ab′)₂ fragments canbe isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In other embodiments, the antibody of choiceis a single-chain Fv fragment (scFv). See, for example, WO 93/16185. Incertain embodiments, the antibody is not a Fab fragment.

5.10. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the ICOS expressing T cell surfacemarker. Other such antibodies may bind a first ICOS expressing T cellmarker and further bind a second ICOS expressing T cell surface marker.An anti-ICOS expressing T cell marker binding arm may also be combinedwith an arm which binds to a triggering molecule on a leukocyte such asa T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG(FcγR), so as to focus cellular defense mechanisms to the ICOSexpressing T cell. Bispecific antibodies may also be used to localizecytotoxic agents to the ICOS expressing T cell. These antibodies possessa ICOS expressing T cell marker-binding arm and an arm which binds thecytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricinA chain, methola-exate or radioactive isotope hapten). Bispecificantibodies can be prepared as full-length antibodies or antibodyfragments (e.g., F(ab′): bispecific antibodies).

Methods for making bispecific antibodies are known in the art. (See, forexample, Millstein et al., Nature, 305:537-539 (1983); Traunecker etal., EMBO J., 10:3655-3659 (1991); Suresh et al., Methods in Enzymology,121:210 (1986); Kostelny et al., J. Immunol., 148(5):1547-1553 (1992);Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993);Gruber et al., J. Immunol., 152:5368 (1994); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980; and4,676,980, WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO92/08802; and EP 03089.)

In one embodiment, where an anti-ICOS antibody of compositions andmethods of the invention is bispecific, the anti-ICOS antibody may behuman or humanized and may have specificity for human ICOS and anepitope on a T cell or may be capable of binding to a human effectorcell such as, for example, a monocyte/macrophage and/or a natural killercell to effect cell death.

5.11. Variant Fc Regions

The present invention provides formulation of proteins comprising avariant Fc region. That is, a non naturally occurring Fc region, forexample an Fc region comprising one or more non naturally occurringamino acid residues. Also encompassed by the variant Fc regions ofpresent invention are Fc regions which comprise amino acid deletions,additions and/or modifications.

It will be understood that Fc region as used herein includes thepolypeptides comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.). The“EU index as set forth in Kabat” refers to the residue numbering of thehuman IgG1 EU antibody as described in Kabat et al. supra. Fc may referto this region in isolation, or this region in the context of anantibody, antibody fragment, or Fc fusion protein. An Fc variant proteinmay be an antibody, Fc fusion, or any protein or protein domain thatcomprises an Fc region including, but not limited to, proteinscomprising variant Fc regions, which are non naturally occurringvariants of an Fc. Note: Polymorphisms have been observed at a number ofFc positions, including but not limited to Kabat 270, 272, 312, 315,356, and 358, and thus slight differences between the presented sequenceand sequences in the prior art may exist.

The present invention encompasses Fc variant proteins which have alteredbinding properties for an Fc ligand (e.g., an Fc receptor, C1q) relativeto a comparable molecule (e.g., a protein having the same amino acidsequence except having a wild type Fc region). Examples of bindingproperties include but are not limited to, binding specificity,equilibrium dissociation constant (K_(D)), dissociation and associationrates (k_(off) and k_(on) respectively), binding affinity and/oravidity. It is generally understood that a binding molecule (e.g., a Fcvariant protein such as an antibody) with a low K_(D) may be preferableto a binding molecule with a high K_(D). However, in some instances thevalue of the k_(on) or k_(off) may be more relevant than the value ofthe K_(D). One skilled in the art can determine which kinetic parameteris most important for a given antibody application.

The affinities and binding properties of an Fc domain for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics(e.g., BIACORE® analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

In one embodiment, the Fc variant protein has enhanced binding to one ormore Fc ligand relative to a comparable molecule. In another embodiment,the Fc variant protein has an affinity for an Fc ligand that is at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold greater than that of a comparable molecule. In a specificembodiment, the Fc variant protein has enhanced binding to an Fcreceptor. In another specific embodiment, the Fc variant protein hasenhanced binding to the Fc receptor FcγRIIIA. In still another specificembodiment, the Fc variant protein has enhanced binding to the Fcreceptor FcRn. In yet another specific embodiment, the Fc variantprotein has enhanced binding to C1q relative to a comparable molecule.

The serum half-life of proteins comprising Fc regions may be increasedby increasing the binding affinity of the Fc region for FcRn. In oneembodiment, the Fc variant protein has enhanced serum half life relativeto comparable molecule.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enables these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. Specific high-affinity IgGantibodies directed to the surface of target cells “arm” the cytotoxiccells and are absolutely required for such killing. Lysis of the targetcell is extracellular, requires direct cell-to-cell contact, and doesnot involve complement. It is contemplated that, in addition toantibodies, other proteins comprising Fc regions, specifically Fc fusionproteins, having the capacity to bind specifically to an antigen-bearingtarget cell will be able to effect cell-mediated cytotoxicity. Forsimplicity, the cell-mediated cytotoxicity resulting from the activityof an Fc fusion protein is also referred to herein as ADCC activity.

The ability of any particular Fc variant protein to mediate lysis of thetarget cell by ADCC can be assayed. To assess ADCC activity an Fcvariant protein of interest is added to target cells in combination withimmune effector cells, which may be activated by the antigen antibodycomplexes resulting in cytolysis of the target cell. Cytolysis isgenerally detected by the release of label (e.g. radioactive substrates,fluorescent dyes or natural intracellular proteins) from the lysedcells. Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361;Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al.,1995 J Immunol Methods 184:29-38. ADCC activity of the Fc variantprotein of interest may also be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad.Sci. USA 95:652-656.

In one embodiment, an Fc variant protein has enhanced ADCC activityrelative to a comparable molecule. In a specific embodiment, an Fcvariant protein has ADCC activity that is at least 2 fold, or at least 3fold, or at least 5 fold or at least 10 fold or at least 50 fold or atleast 100 fold greater than that of a comparable molecule. In anotherspecific embodiment, an Fc variant protein has enhanced binding to theFc receptor FcγRIIIA and has enhanced ADCC activity relative to acomparable molecule. In other embodiments, the Fc variant protein hasboth enhanced ADCC activity and enhanced serum half life relative to acomparable molecule.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget cell in the presence of complement. The complement activationpathway is initiated by the binding of the first component of thecomplement system (C1q) to a molecule, an antibody for example,complexed with a cognate antigen. To assess complement activation, a CDCassay, e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol.Methods, 202:163, may be performed. In one embodiment, an Fc variantprotein has enhanced CDC activity relative to a comparable molecule. Ina specific embodiment, an Fc variant protein has CDC activity that is atleast 2 fold, or at least 3 fold, or at least 5 fold or at least 10 foldor at least 50 fold or at least 100 fold greater than that of acomparable molecule. In other embodiments, the Fc variant protein hasboth enhanced CDC activity and enhanced serum half life relative to acomparable molecule.

In one embodiment, the present invention provides compositions, whereinthe Fc region comprises a non naturally occurring amino acid residue atone or more positions selected from the group consisting of 234, 235,236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255,256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296,297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 332, 333,334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 asnumbered by the EU index as set forth in Kabat. Optionally, the Fcregion may comprise a non naturally occurring amino acid residue atadditional and/or alternative positions known to one skilled in the art(see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

In a specific embodiment, the present invention provides an Fc variantprotein composition, wherein the Fc region comprises at least one nonnaturally occurring amino acid residue selected from the groupconsisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V,234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y,235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y,240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R, 243W, 243L 243Y,243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E,256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W,264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V,265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N,296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T,298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D,325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I,330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H,332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Yand 434W as numbered by the EU index as set forth in Kabat. Optionally,the Fc region may comprise additional and/or alternative non naturallyoccurring amino acid residues known to one skilled in the art (see,e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT PatentPublications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752 and WO 05/040217).

In another embodiment, the present invention provides an Fc variantprotein composition, wherein the Fc region comprises at least a nonnaturally occurring amino acid at one or more positions selected fromthe group consisting of 239, 330 and 332, as numbered by the EU index asset forth in Kabat. In a specific embodiment, the present inventionprovides an Fc variant protein formulation, wherein the Fc regioncomprises at least one non naturally occurring amino acid selected fromthe group consisting of 239D, 330L and 332E, as numbered by the EU indexas set forth in Kabat. Optionally, the Fc region may further compriseadditional non naturally occurring amino acid at one or more positionsselected from the group consisting of 252, 254, and 256, as numbered bythe EU index as set forth in Kabat. In a specific embodiment, thepresent invention provides an Fc variant protein formulation, whereinthe Fc region comprises at least one non naturally occurring amino acidselected from the group consisting of 239D, 330L and 332E, as numberedby the EU index as set forth in Kabat and at least one non naturallyoccurring amino acid at one or more positions are selected from thegroup consisting of 252Y, 254T and 256E, as numbered by the EU index asset forth in Kabat.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Ghetieet al., 1997, Nat. Biotech. 15:637-40; Duncan et al, 1988, Nature332:563-564; Lund et al., 1991, J. Immunol. 147:2657-2662; Lund et al,1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351. Also encompassed by the present invention are Fc regionswhich comprise deletions, additions and/or modifications. Still othermodifications/substitutions/additions/deletions of the Fc domain will bereadily apparent to one skilled in the art.

Methods for generating non naturally occurring Fc regions are known inthe art. For example, amino acid substitutions and/or deletions can begenerated by mutagenesis methods, including, but not limited to,site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492(1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methodsand Applications”, Academic Press, San Diego, pp. 177-183 (1990)), andcassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably,site-directed mutagenesis is performed by the overlap-extension PCRmethod (Higuchi, in “PCR Technology: Principles and Applications for DNAAmplification”, Stockton Press, New York, pp. 61-70 (1989)). Thetechnique of overlap-extension PCR (Higuchi, ibid.) can also be used tointroduce any desired mutation(s) into a target sequence (the startingDNA). For example, the first round of PCR in the overlap-extensionmethod involves amplifying the target sequence with an outside primer(primer 1) and an internal mutagenesis primer (primer 3), and separatelywith a second outside primer (primer 4) and an internal primer (primer2), yielding two PCR segments (segments A and B). The internalmutagenesis primer (primer 3) is designed to contain mismatches to thetarget sequence specifying the desired mutation(s). In the second roundof PCR, the products of the first round of PCR (segments A and B) areamplified by PCR using the two outside primers (primers 1 and 4). Theresulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector. As the first step of mutagenesis, thestarting DNA (e.g., encoding an Fc fusion protein, an antibody or simplyan Fc region), is operably cloned into a mutagenesis vector. The primersare designed to reflect the desired amino acid substitution. Othermethods useful for the generation of variant Fc regions are known in theart (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. PatentPublication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO04/063351).

In some embodiments, an Fc variant protein comprises one or moreengineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to the molecule comprising an Fc region. Engineeredglycoforms may be useful for a variety of purposes, including but notlimited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton,N.J.); GlycoMAb™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739;EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

5.12. Glycosylation of Antibodies

In still another embodiment, the glycosylation of antibodies utilized inaccordance with the invention is modified. For example, an aglycoslatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody for a target antigen. Such carbohydrate modifications canbe accomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861. One or moreamino acid substitutions can also be made that result in elimination ofa glycosylation site present in the Fc region (e.g., Asparagine 297 ofIgG). Furthermore, aglycosylated antibodies may be produced in bacterialcells which lack the necessary glycosylation machinery.

An antibody can also be made that has an altered type of glycosylation,such as a hypofucosylated antibody having reduced amounts of fucosylresidues or an antibody having increased bisecting GlcNAc structures.Such altered glycosylation patterns have been demonstrated to increasethe ADCC ability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, U.S. Pat. No. 6,946,292;European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO99/54342 each of which is incorporated herein by reference in itsentirety.

Antibodies with altered glycosylation pattern may also be generatedusing lower eukaryotic host cells comprising a modified glycosylationmachinery as described in U.S. Pat. No. 7,029,872, US Patent PublicationUS20060148035A1, each of which is incorporated herein by reference inits entirety.

5.13. Engineering Effector Function

It may be desirable to modify an anti-ICOS antibody of the inventionwith respect to effector function, so as to enhance the effectiveness ofthe antibody in treating T cell-mediated diseases, for example. Forexample, cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing and/orantibody-dependent cellular cytotoxicity (ADCC) and/or antibodydependent phagocytosis. See, Caron et al., J. Exp Med., 176:1191-1195(1992) and Shopes, B., J. Immunol., 148:2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al, CancerResearch, 53:2560-2565 (1993). An antibody can also be engineered whichhas dual Fc regions and may thereby have enhanced complement lysis,antibody-dependent phagocytosis and/or ADCC capabilities. See, Stevensonet al., Anti-Cancer Drug Design, 3:219-230 (1989).

Other methods of engineering Fc regions of antibodies so as to altereffector functions are known in the art (e.g., U.S. Patent PublicationNo. 20040185045 and PCT Publication No. WO 2004/016750, both to Koeniget al., which describe altering the Fc region to enhance the bindingaffinity for FcγRIIB as compared with the binding affinity for FCγRIIA;see, also, PCT Publication Nos. WO 99/58572 to Armour et al., WO99/51642 to Idusogie et al., and U.S. Pat. No. 6,395,272 to Deo et al.;the disclosures of which are incorporated herein in their entireties).Methods of modifying the Fc region to decrease binding affinity toFcγRIIB are also known in the art (e.g., U.S. Patent Publication No.20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.,the disclosures of which are incorporated herein in their entireties).Modified antibodies having variant Fc regions with enhanced bindingaffinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fcregion have also been described (e.g., PCT Publication Nos. WO2004/063351, to Stavenhagen et al., the disclosure of which isincorporated herein in its entirety).

In vitro assays known in the art can be used to determine whetheranti-ICOS antibodies used in compositions and methods of the inventionare capable of mediating ADCC, CDC, and/or antibody-dependentphagocytosis, such as those described herein.

5.14. Manufacture/Production of Anti-ICOS Antibodies

Once a desired anti-ICOS antibody is engineered, the anti-ICOS antibodycan be produced on a commercial scale using methods that are well-knownin the art for large scale manufacturing of antibodies. For example,this can be accomplished using recombinant expressing systems such as,but not limited to, those described below.

5.15. Recombinant Expression Systems

Recombinant expression of an antibody or variant thereof, generallyrequires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof, has been obtained, the vector for the production of theantibody molecule may be produced by recombinant DNA technology usingtechniques well-known in the art. See, e.g., U.S. Pat. No. 6,331,415,which is incorporated herein by reference in its entirety. Thus, methodsfor preparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well-known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule, a heavy or light chain of an antibody, aheavy or light chain variable domain of an antibody or a portionthereof, or a heavy or light chain CDR, operably linked to a promoter.Such vectors may include the nucleotide sequence encoding the constantregion of the antibody molecule (see, e.g., International PublicationNos. WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464) and thevariable domain of the antibody may be cloned into such a vector forexpression of the entire heavy, the entire light chain, or both theentire heavy and light chains.

In another embodiment, anti-ICOS antibodies can be made using targetedhomologous recombination to produce all or portions of the anti-ICOSantibodies (see, U.S. Pat. Nos. 6,063,630, 6,187,305, and 6,692,737). Incertain embodiments, anti-ICOS antibodies can be made using randomrecombination techniques to produce all or portions of the anti-ICOSantibodies (see, U.S. Pat. Nos. 6,361,972, 6,524,818, 6,541,221, and6,623,958). Anti-ICOS antibodies can also be produced in cellsexpressing an antibody from a genomic sequence of the cell comprising amodified immunoglobulin locus using Cre-mediated site-specifichomologous recombination (see, U.S. Pat. No. 6,091,001). The host cellline may be derived from human or nonhuman species including but notlimited to mouse, and Chinese hamster. Where human or humanized antibodyproduction is desired, the host cell line should be a human cell line.These methods may advantageously be used to engineer stable cell lineswhich permanently express the antibody molecule.

Once the expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding an antibody of the inventionor fragments thereof, or a heavy or light chain thereof, or portionthereof, or a single-chain antibody of the invention, operably linked toa heterologous promoter. In certain embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressan anti-ICOS antibody or portions thereof that can be used in theengineering and generation of anti-ICOS antibodies (see, e.g., U.S. Pat.No. 5,807,715). For example, mammalian cells such as Chinese hamsterovary cells (CHO), in conjunction with a vector such as the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for antibodies (Foecking et al., Gene,45:101 (1986); and Cockett et al., Bio/Technology, 8:2 (1990)). Inaddition, a host cell strain may be chosen which modulates theexpression of inserted antibody sequences, or modifies and processes theantibody gene product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the antibody or portionthereof expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, WI 38, BT483, Hs578T, HTB2, BT2O and T47D,NS0 (a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), CRL7O3O and HsS78Bst cells.

In one embodiment, human cell lines developed by immortalizing humanlymphocytes can be used to recombinantly produce monoclonal humananti-ICOS antibodies.

In one embodiment, the human cell line PER.C6. (Crucell, Netherlands)can be used to recombinantly produce monoclonal human anti-ICOSantibodies.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions comprising an anti-ICOS antibody, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J.Biol. Chem., 24:5503-5509 (1989)); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione-S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to glutathione-agarose affinity matrix followed by elution inthe presence of free glutathione. The pGEX vectors are designed tointroduce athrombin and/or factor Xa protease cleavage sites into theexpressed polypeptide so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example, the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter).

In mammalian host cells, a number of virus based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion into a non-essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts (e.g., see, Logan& Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon should generally be in frame with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, e.g., Bittneret al., Methods in Enzymol., 153:51-544 (1987)).

Stable expression can be used for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express theantibody molecule may be generated. Host cells can be transformed withan appropriately engineered vector comprising expression controlelements (e.g., promoter, enhancer, transcription terminators,polyadenylation sites, etc.), and a selectable marker gene. Followingthe introduction of the foreign DNA, cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells that stably integrated theplasmid into their chromosomes to grow and form foci which in turn canbe cloned and expanded into cell lines. Plasmids that encode ananti-ICOS antibody can be used to introduce the gene/cDNA into any cellline suitable for production in culture.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell,11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes canbe employed in tk⁻, hgprt⁻ or aprT⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH11(5):155-2 15 (1993)); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981,J. Mol. Biol., 150:1, which are incorporated by reference herein intheir entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see, Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. Academic Press, New York(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol., 3:257(1983)). Antibody expression levels may be amplified through the userecombinant methods and tools known to those skilled in the art ofrecombinant protein production, including technologies that remodelsurrounding chromatin and enhance transgene expression in the form of anactive artificial transcriptional domain.

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical or different selectable markers. A single vector whichencodes, and is capable of expressing, both heavy and light chainpolypeptides may also be used. In such situations, the light chainshould be placed 5′ to the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature 322:562-65 (1986); and Kohler, 1980,Proc. Natl. Acad. Sci. USA, 77:2197 (1980)). The coding sequences forthe heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule has been produced by recombinant expression,it may be purified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigensProtein A or Protein G, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. Further, the antibodies ofthe present invention or fragments thereof may be fused to heterologouspolypeptide sequences described herein or otherwise known in the art tofacilitate purification.

5.15.1. Antibody Purification and Isolation

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology, 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted into the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibodymutant is secreted into the medium, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or MilliporePellicon ultrafiltration unit. A protease inhibitor such as PMSF may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, ion exchange chromatography, gel electrophoresis,dialysis, and/or affinity chromatography either alone or in combinationwith other purification steps. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody mutant. Protein A can be usedto purify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods, 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ., 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH₃ domain, the Bakerbond ABX resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin, SEPHAROSE chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, and performed at low salt concentrations (e.g.,from about 0-0.25 M salt).

5.16. Therapeutic Anti-ICOS Antibodies

An anti-ICOS antibody used in compositions and methods of the inventionmay be a human antibody or a humanized antibody that may mediate T celllineage ADCC, antibody-dependent phagocytosis and/or CDC, or can beselected from known anti-ICOS antibodies that may mediate T lineage cellADCC, antibody-dependent phagocytosis and/or CDC. In certainembodiments, anti-ICOS antibodies can be chimeric antibodies. In certainembodiments, an anti-ICOS antibody can be a monoclonal human, humanized,or chimeric anti-ICOS antibody. An anti-ICOS antibody used incompositions and methods of the invention may be a human antibody or ahumanized antibody of the IgG1 or IgG3 human isotype or any IgG1 or IgG3allele found in the human population. In other embodiments, an anti-ICOSantibody used in compositions and methods of the invention can be ahuman antibody or a humanized antibody of the IgG2 or IgG4 human isotypeor any IgG2 or IgG4 allele found in the human population.

While such antibodies can be generated using the techniques describedabove, in other embodiments of the invention, the human JMab-136anti-ICOS antibody (see, U.S. Pat. No. 6,803,039) can be modified togenerate an anti-ICOS antibody with enhanced effector function such as,but not limited to, ADCC, antibody-dependent phagocytosis and/or CDC.For example, known anti-ICOS antibodies that can be used include, butare not limited to, anti-human ICOS monoclonal antibodies disclosed inU.S. Pat. No. 6,803,039, and clone ISA-3 (eBioscience, US).

In certain embodiments, the antibody is an isotype switched variant of aknown antibody (e.g., to an IgG1 or IgG3 human isotype) such as thosedescribed above.

An anti-ICOS antibodies used in compositions and methods of theinvention can be naked antibodies, immunoconjugates or fusion proteins.Anti-ICOS antibodies described above for use in compositions and methodsof the invention may be able to reduce or deplete ICOS expressing Tcells and circulating immunoglobulin in a human treated therewith.Depletion of T cells can be in circulating T cells, or in particulartissues such as, but not limited to, bone marrow, spleen, gut-associatedlymphoid tissues, and/or lymph nodes. Such depletion may be achieved viavarious mechanisms such as antibody-dependent cell-mediated cytotoxicity(ADCC), and/or antibody dependent phagocytosis, and/or by blocking ofICOS interaction with its intended ligand, and/or complement dependentcytotoxicity (CDC). By “depletion” of T cells it is meant a reduction incirculating ICOS expressing T cells and/or ICOS expressing T cells inparticular tissue(s) by at least about 25%, 40%, 50%, 65%, 75%, 80%,85%, 90%, 95% or more. In particular embodiments, virtually alldetectable ICOS expressing T cells are depleted from the circulationand/or particular tissue(s). By “depletion” of circulatingimmunoglobulin (Ig) it is meant a reduction by at least about 25%, 40%,50%, 65%, 75%, 80%, 85%, 90%, 95% or more. In particular embodiments,virtually all detectable Ig is depleted from the circulation.

5.16.1. Screening of Antibodies for Human ICOS Binding

Binding assays can be used to identify antibodies that bind the humanICOS antigen. Binding assays may be performed either as direct bindingassays or as competition-binding assays. Binding can be detected usingstandard ELISA or standard Flow Cytometry assays. In a direct bindingassay, a candidate antibody is tested for binding to human ICOS antigen.In certain embodiments, the screening assays comprise, in a second step,determining the ability to of an antibody to induce downstream signalingevents in T cells expressing human ICOS. Competition-binding assays, onthe other hand, assess the ability of a candidate antibody to competewith a known anti-ICOS antibody or other compound that binds human ICOS.

In a direct binding assay, the human ICOS antigen is contacted with acandidate antibody under conditions that allow binding of the candidateantibody to the human ICOS antigen. The binding may take place insolution or on a solid surface. The candidate antibody may have beenpreviously labeled for detection. Any detectable compound can be usedfor labeling, such as, but not limited to, a luminescent, fluorescent,or radioactive isotope or group containing same, or a nonisotopic label,such as an enzyme or dye. After a period of incubation sufficient forbinding to take place, the reaction is exposed to conditions andmanipulations that remove excess or non-specifically bound antibody.Typically, it involves washing with an appropriate buffer. Finally, thepresence of a ICOS-antibody complex is detected.

In a competition-binding assay, a candidate antibody is evaluated forits ability to inhibit or displace the binding of a known anti-ICOSantibody (or other compound) to the human ICOS antigen. A labeled knownbinder of ICOS may be mixed with the candidate antibody, and placedunder conditions in which the interaction between them would normallyoccur, with and without the addition of the candidate antibody. Theamount of labeled known binder of ICOS that binds the human ICOS may becompared to the amount bound in the presence or absence of the candidateantibody.

In one embodiment, the binding assay is carried out with one or morecomponents immobilized on a solid surface to facilitate antibody antigencomplex formation and detection. In various embodiments, the solidsupport could be, but is not restricted to, polyvinylidene fluoride,polycarbonate, polystyrene, polypropylene, polyethylene, glass,nitrocellulose, dextran, nylon, polyacrylamide and agarose. The supportconfiguration can include beads, membranes, microparticles, the interiorsurface of a reaction vessel such as a microtiter plate, test tube orother reaction vessel. The immobilization of human ICOS, or othercomponent, can be achieved through covalent or non-covalent attachments.In one embodiment, the attachment may be indirect, i.e., through anattached antibody. In another embodiment, the human ICOS antigen andnegative controls are tagged with an epitope, such as glutathioneS-transferase (GST) so that the attachment to the solid surface can bemediated by a commercially available antibody such as anti-GST (SantaCruz Biotechnology).

For example, such an affinity binding assay may be performed using thehuman ICOS antigen which is immobilized to a solid support. Typically,the non-mobilized component of the binding reaction, in this case thecandidate anti-ICOS antibody, is labeled to enable detection. A varietyof labeling methods are available and may be used, such as luminescent,chromophore, fluorescent, or radioactive isotope or group containingsame, and nonisotopic labels, such as enzymes or dyes. In oneembodiment, the candidate anti-ICOS antibody is labeled with afluorophore such as fluorescein isothiocyanate (FITC, available fromSigma Chemicals, St. Louis). Such an affinity binding assay may beperformed using the human ICOS antigen immobilized on a solid surface.Anti-ICOS antibodies are then incubated with the antigen and thespecific binding of antibodies is detected by methods known in the artincluding, but not limited to, BiaCore Analyses, ELISA, FMET and RIAmethods.

Finally, the label remaining on the solid surface may be detected by anydetection method known in the art. For example, if the candidateanti-ICOS antibody is labeled with a fluorophore, a fluorimeter may beused to detect complexes.

The human ICOS antigen can be added to binding assays in the form ofintact cells that express human ICOS antigen, or isolated membranescontaining human ICOS antigen. Thus, direct binding to human ICOSantigen may be assayed in intact cells in culture or in animal models inthe presence and absence of the candidate anti-ICOS antibody. A labeledcandidate anti-ICOS antibody may be mixed with cells that express humanICOS antigen, or with crude extracts obtained from such cells, and thecandidate anti-ICOS antibody may be added. Isolated membranes may beused to identify candidate anti-ICOS antibodies that interact with humanICOS. For example, in a typical experiment using isolated membranes,cells may be genetically engineered to express human ICOS antigen.Membranes can be harvested by standard techniques and used in an invitro binding assay. Labeled candidate anti-ICOS antibody (e.g.,fluorescent labeled antibody) is bound to the membranes and assayed forspecific activity; specific binding is determined by comparison withbinding assays performed in the presence of excess unlabeled (cold)candidate anti-ICOS antibody. Soluble human ICOS antigen may also berecombinantly expressed and utilized in non-cell based assays toidentify antibodies that bind to human ICOS antigen. The recombinantlyexpressed human ICOS polypeptides can be used in the non-cell basedscreening assays. Peptides corresponding to one or more of the bindingportions of human ICOS antigen, or fusion proteins containing one ormore of the binding portions of human ICOS antigen can also be used innon-cell based assay systems to identify antibodies that bind toportions of human ICOS antigen. In non-cell based assays therecombinantly expressed human ICOS is attached to a solid substrate suchas a test tube, microtiter well or a column, by means well-known tothose in the art (see, Ausubel et al., supra). The test antibodies arethen assayed for their ability to bind to human ICOS antigen.

The binding reaction may also be carried out in solution. In this assay,the labeled component is allowed to interact with its binding partner(s)in solution. If the size differences between the labeled component andits binding partner(s) permit such a separation, the separation can beachieved by passing the products of the binding reaction through anultrafilter whose pores allow passage of unbound labeled component butnot of its binding partner(s) or of labeled component bound to itspartner(s). Separation can also be achieved using any reagent capable ofcapturing a binding partner of the labeled component from solution, suchas an antibody against the binding partner and so on.

In another specific embodiment, the solid support is membrane containinghuman ICOS antigen attached to a microtiter dish. Candidate antibodies,for example, can bind cells that express library antibodies cultivatedunder conditions that allow expression of the library members in themicrotiter dish. Library members that bind to the human ICOS areharvested. Such methods, are generally described by way of example inParmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al., 1992,BioTechniques, 13:422-427; PCT Publication No. WO94/18318; and inreferences cited hereinabove. Antibodies identified as binding to humanICOS antigen can be of any of the types or modifications of antibodiesdescribed above.

5.16.2. Screening of Antibodies for Human ADCC Effector Function

Antibodies of the human IgG class, which have functional characteristicssuch a long half-life in serum and the ability to mediate variouseffector functions are used in certain embodiments of the invention(Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc.,Chapter 1 (1995)). The human IgG class antibody is further classifiedinto the following 4 subclasses: IgG1, IgG2, IgG3 and IgG4. A largenumber of studies have so far been conducted for ADCC and CDC aseffector functions of the IgG class antibody, and it has been reportedthat among antibodies of the human IgG class, the IgG1 subclass has thehighest ADCC activity and CDC activity in humans (Chemical Immunology,65, 88 (1997)).

Expression of ADCC activity and CDC activity of the human IgG1 subclassantibodies generally involves binding of the Fc region of the antibodyto a receptor for an antibody (hereinafter referred to as “FcγR”)existing on the surface of effector cells such as killer cells, naturalkiller cells or activated macrophages. Various complement components canbe bound. Regarding the binding, it has been suggested that severalamino acid residues in the hinge region and the second domain of Cregion (hereinafter referred to as “Cγ2 domain”) of the antibody areimportant (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319(1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain inthe Cγ2 domain (Chemical Immunology, 65, 88 (1997)) is also important.

Anti-ICOS antibodies can be modified with respect to effector function,e.g., so as to enhance ADCC and/or complement dependent cytotoxicity(CDC) of the antibody. This may be achieved by introducing one or moreamino acid substitutions in the Fc region of an antibody. Cysteineresidue(s) may also be introduced in the Fc region, allowing forinterchain disulfide bond formation in this region. In this way ahomodimeric antibody can be generated that may have improvedinternalization capability and or increased complement-mediated cellkilling and ADCC (Caron et al., J. Exp. Med., 176:1191-1195 (1992) andShopes, J. Immunol., 148:2918-2922 (1992)). Heterobifunctionalcross-linkers can also be used to generate homodimeric antibodies withenhanced anti-tumor activity (Wolff et al, Cancer Research, 53:2560-2565(1993)). Antibodies can also be engineered to have two or more Fcregions resulting in enhanced complement lysis and ADCC capabilities(Stevenson et al., Anti-Cancer Drug Design, (3)219-230 (1989)).

Other methods of engineering Fc regions of antibodies so as to altereffector functions are known in the art (e.g., U.S. Patent PublicationNo. 20040185045 and PCT Publication No. WO 2004/016750, both to Koeniget al., which describe altering the Fc region to enhance the bindingaffinity for FcγRIB as compared with the binding affinity for FCγRIIA;see also PCT Publication Nos. WO 99/58572 to Armour et al., WO 99/51642to Idusogie et al., and U.S. Pat. No. 6,395,272 to Deo et al.; thedisclosures of which are incorporated herein in their entireties).Methods of modifying the Fc region to decrease binding affinity toFcγRIB are also known in the art (e.g., U.S. Patent Publication No.20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.,the disclosures of which are incorporated herein in their entireties).Modified antibodies having variant Fc regions with enhanced bindingaffinity for FcγRIIIA and/or FcγRIIA as compared with a wildtype Fcregion have also been described (e.g., PCT Publication No. WO2004/063351, to Stavenhagen et al.; the disclosure of which isincorporated herein in its entirety).

At least four different types of FcγR have been found, which arerespectively called FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), andFcγRIV. In human, FcγRII and FcγRIII are further classified into FcγRIIaand FcγRIIb, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membraneprotein belonging to the immunoglobulin superfamily, FcγRII, FcγRIII,and FcγRIV have an a chain having an extracellular region containing twoimmunoglobulin-like domains, FcγRI has an a chain having anextracellular region containing three immunoglobulin-like domains, as aconstituting component, and the α chain is involved in the IgG bindingactivity. In addition, FcγRI and FcγRIII have a γ chain or ζ chain as aconstituting component which has a signal transduction function inassociation with the a chain (Annu. Rev. Immunol., 18, 709 (2000), Annu.Rev. Immunol., 19, 275 (2001)). FcγRIV has been described by Bruhns etal., Clin. Invest. Med., (Canada) 27:3 D (2004).

To assess ADCC activity of an anti-ICOS antibody of interest, an invitro ADCC assay can be used, such as that described in U.S. Pat. No.5,500,362 or 5,821,337. The assay may also be performed using acommercially available kit, e.g. CytoTox 96® (Promega). Useful effectorcells for such assays include, but are not limited to peripheral bloodmononuclear cells (PBMC), Natural Killer (NK) cells, and NK cell lines.NK cell lines expressing a transgenic Fc receptor (e.g. CD16) andassociated signaling polypeptide (e.g. FCεRI-γ) may also serve aseffector cells (see, e.g. WO 2006/023148 A2 to Campbell). For example,the ability of any particular antibody to mediate lysis of the targetcell by complement activation and/or ADCC can be assayed. The cells ofinterest are grown and labeled in vitro; the antibody is added to thecell culture in combination with immune cells which may be activated bythe antigen antibody complexes; i.e., effector cells involved in theADCC response. The antibody can also be tested for complementactivation. In either case, cytolysis of the target cells is detected bythe release of label from the lysed cells. The extent of target celllysis may also be determined by detecting the release of cytoplasmicproteins (e.g. LDH) into the supernatant. In fact, antibodies can bescreened using the patient's own serum as a source of complement and/orimmune cells. The antibodies that are capable of mediating human ADCC inthe in vitro test can then be used therapeutically in that particularpatient. ADCC activity of the molecule of interest may also be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Moreover,techniques for modulating (i.e., increasing or decreasing) the level ofADCC, and optionally CDC activity, of an antibody are well-known in theart. See, e.g., U.S. Pat. No. 6,194,551. Antibodies of the presentinvention may be capable or may have been modified to have the abilityof inducing ADCC and/or CDC. Assays to determine ADCC function can bepracticed using human effector cells to assess human ADCC function. Suchassays may also include those intended to screen for antibodies thatinduce, mediate, enhance, block cell death by necrotic and/or apoptoticmechanisms. Such methods including assays utilizing viable dyes, methodsof detecting and analyzing caspases, and assays measuring DNA breaks canbe used to assess the apoptotic activity of cells cultured in vitro withan anti-ICOS antibody of interest.

For example, Annexin V or TdT-mediated dUTP nick-end labeling (TUNEL)assays can be carried out as described in Decker et al., Blood (USA)103:2718-2725 (2004) to detect apoptotic activity. The TUNEL assayinvolves culturing the cell of interest with fluorescein-labeled dUTPfor incorporation into DNA strand breaks. The cells are then processedfor analysis by flow cytometry. The Annexin V assay detects theappearance of phosphatidylserine (PS) on the outside of the plasmamembrane of apoptotic cells using a fluorescein-conjugated Annexin Vthat specifically recognizes the exposed PS molecules. Concurrently, aviable dye such as propidium iodide can be used to exclude lateapoptotic cells. The cells are stained with the labeled Annexin V andare analyzed by flow cytometry.

5.16.3. Immunoconjugates and Fusion Proteins

According to certain aspects of the invention, therapeutic agents ortoxins can be conjugated to anti-ICOS antibodies for use in compositionsand methods of the invention. In certain embodiments, these conjugatescan be generated as fusion proteins. Examples of therapeutic agents andtoxins include, but are not limited to, members of the enediyne familyof molecules, such as calicheamicin and esperamicin. Chemical toxins canalso be taken from the group consisting of duocarmycin (see, e.g., U.S.Pat. No. 5,703,080 and U.S. Pat. No. 4,923,990), methotrexate,doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C,cis-platinum, etoposide, bleomycin and 5-fluorouracil. Examples ofchemotherapeutic agents also include Adriamycin, Doxorubicin,5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide,Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate,Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide,mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin,Teniposide, Daunomycin, Caminomycin, Aminopterin, Dactinomycin,Mitomycins, Esperamicins (see, U.S. Pat. No. 4,675,187), Melphalan, andother related nitrogen mustards.

In certain embodiments, anti-ICOS antibodies are conjugated to acytostatic, cytotoxic or immunosuppressive agent wherein the cytotoxicagent is selected from the group consisting of an enediyne, alexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, amaytansinoid, and a vinca alkaloid. In certain, more specificembodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065,SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin,calicheamicin, maytansine, DM-1, auristatin E, AEB, AEVB, AEFP, MMAE(see, U.S. patent application Ser. No. 10/983,340), or netropsin.

In certain embodiments, the cytotoxic agent of an anti-ICOSantibody-cytotoxic agent conjugate of the invention is an anti-tubulinagent. In specific embodiments, the cytotoxic agent is selected from thegroup consisting of a vinca alkaloid, a podophyllotoxin, a taxane, abaccatin derivative, a cryptophysin, a maytansinoid, a combretastatin,and a dolastatin. In other embodiments, the cytotoxic agent isvincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin,paclitaxel, docetaxel, epithilone A, epithilone B, nocodazole,coichicine, colcimid, estramustine, cemadotin, discodermolide,maytansine, DM-1, AEFP, auristatin E, AEB, AEVB, AEFP, MMAE oreleutherobin.

In specific embodiments, an anti-ICOS antibody is conjugated to thecytotoxic agent via a linker, wherein the linker is peptide linker. Inother embodiments, an anti-ICOS antibody is conjugated to the cytotoxicagent via a linker, wherein the linker is a val-cit linker, a phe-lyslinker, a hydrazone linker, or a disulfide linker.

In certain embodiments, the anti-ICOS antibody of an anti-ICOSantibody-cytotoxic agent conjugate is conjugated to the cytotoxic agentvia a linker, wherein the linker is hydrolysable at a pH of less than5.5. In a specific embodiment the linker is hydrolyzable at a pH of lessthan 5.0.

In certain embodiments, the anti-ICOS antibody of an anti-ICOSantibody-cytotoxic agent conjugate is conjugated to the cytotoxic agentvia a linker, wherein the linker is cleavable by a protease. In aspecific embodiment, the protease is a lysosomal protease. In otherembodiments, the protease is, inter alia, a membrane-associatedprotease, an intracellular protease, or an endosomal protease.

Other toxins that can be used in immunoconjugates of the inventioninclude poisonous lectins, plant toxins such as ricin, abrin, modeccin,botulina, and diphtheria toxins. Of course, combinations of the varioustoxins could also be coupled to one antibody molecule therebyaccommodating variable cytotoxicity. Illustrative of toxins which aresuitably employed in combination therapies of the invention are ricin,abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweedanti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell, 47:641(1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43(1994). Enzymatically active toxins and fragments thereof which can beused include diphtheria A chain, non-binding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

Suitable toxins and chemotherapeutic agents are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and inGoodman And Gilman's The Pharmacological Basis of Therapeutics, 7th Ed.(MacMillan Publishing Co. 1985). Other suitable toxins and/orchemotherapeutic agents are known to those of skill in the art.

The present invention further encompasses antibodies (including antibodyfragments or variants thereof) comprising or conjugated to a radioactiveagent suitable for diagnostic purposes. Examples of suitable radioactivematerials include, but are not limited to, iodine (¹²¹I, ¹²³I, ¹²⁵I,¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In,^(113m)In, ^(115m)In), technetium (⁹⁹Tc, ⁹⁹ mTc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³⁵Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴ Pm, ¹⁴La, ¹⁷⁵Yb, ¹⁶⁶Ho,⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

Further, an anti-ICOS antibody of the invention (including an scFv orother molecule comprising, or alternatively consisting of, antibodyfragments or variants thereof), may be coupled or conjugated to aradioactive metal ion utilized for therapeutic purposes. Examples ofsuitable radioactive ions include, but are not limited to,alpha-emitters such as ²¹³Bi, or other radioisotopes such as ¹⁰³Pd,¹³⁵Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb,⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³ Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re, ¹⁸⁸Re and ¹⁶⁶Ho. Inspecific embodiments, an antibody or fragment thereof is attached tomacrocyclic chelators that chelate radiometal ions, including but notlimited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In specificembodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the an antibody ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes 4(10):2483-90, 1998; Peterson et al., Bioconjug Chem 10(4):553-7,1999; and Zimmerman et al., Nucl Med Biol 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety.

An anti-ICOS antibody of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g., a peptidyl chemotherapeutic agent, see,WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278. The enzyme component of the immunoconjugateuseful for ADEPT includes any enzyme capable of acting on a prodrug insuch a way so as to covert it into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with α-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Antibodies with enzymatic activity, also known in the art as“abzymes,” can be used as well to convert the prodrugs into free activedrugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme as desired to portions of a human affected by a ICOS expressing Tcell malignancy.

Antibodies of this invention may be covalently bound to the enzymes bytechniques well-known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Fusionproteins comprising at least the antigen-binding region of an anti-ICOSantibody linked to at least a functionally active portion of an enzymemay also be constructed using recombinant DNA techniques well-known inthe art (see, e.g., Neuberger et al., Nature, 312:604-608 (1984)).

Covalent modifications of an anti-ICOS antibody are included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of an anti-ICOS antibody are introducedinto the molecule by reacting targeted amino acid residues of theantibody with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Similarly,iodo-reagents may also be used. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction canbe performed in 0.1 M sodium cacodylate at pH 6.0.

Lysyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues and/orε-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residuesgenerally requires that the reaction be performed in alkaline conditionsbecause of the high pKa of the guanidine functional group. Furthermore,these reagents may react with the ε-amino groups of lysine as well asthe arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl—4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

5.17. Chemotherapeutic Combinations

According to the invention, cancer or one or more symptoms thereof maybe prevented, treated, managed or ameliorated by the administration ofan anti-ICOS mAb in combination with the administration of one or moretherapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies.

In a specific embodiment, methods of the invention encompass theadministration of one or more angiogenesis antagonists such as but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); Fibronectin fragment; Gro-beta; Halofuginone;Heparinases; Heparin hexasaccharide fragment; HMV833; Human chorionicgonadotropin (hCG); IM-862; Interferon alpha/beta/gamma; Interferoninducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogenfragment); Marimastat; Metalloproteinase inhibitors (TIMPs);2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-IC11; Neovastat;NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Plasminogenactivator inhibitor; Platelet factor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-related protein (PRP); PTK 787/ZK 222594;Retinoids; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-b);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD 6474;farnesyl transferase inhibitors (FTI); and bisphosphonates (such as butare not limited to, alendronate, clodronate, etidronate, ibandronate,pamidronate, risedronate, tiludronate, and zoledronate).

In a specific embodiment, methods of the invention encompass theadministration of one or more immunomodulatory agents, such as but notlimited to, chemotherapeutic agents and non-chemotherapeuticimmunomodulatory agents. Non-limiting examples of chemotherapeuticagents include methotrexate, cyclosporin A, leflunomide, cisplatin,ifosfamide, taxanes such as taxol and paclitaxol, topoisomerase Iinhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine,vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin,vinorelbine, temodal, cytochalasin B, gramicidin D, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin homologues, andcytoxan. Examples of non-chemotherapeutic immunomodulatory agentsinclude, but are not limited to, anti-T cell receptor antibodies (e.g.,anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC andSKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson &Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380(Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies(e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)),anti-CD2 antibodies (e.g., MEDI-507 (MedImmune, Inc., InternationalPublication Nos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies(e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114)(IDEC)); anti-cytokine receptor antibodies (e.g., anti-IFN receptorantibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein DesignLabs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies,anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies),anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-αantibodies, anti-IL-1β antibodies, anti-IL-6 antibodies, anti-IL-8antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-12 antibodies andanti-IL-23 antibodies)); CTLA4-immunoglobulin; LFA-3TIP (Biogen,International Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432);soluble cytokine receptors (e.g., the extracellular domain of a TNF-αreceptor or a fragment thereof, the extracellular domain of an IL-1βreceptor or a fragment thereof, and the extracellular domain of an IL-6receptor or a fragment thereof); cytokines or fragments thereof (e.g.,interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-15, IL-23, TNF-α, TNF-β, interferon (IFN)-α, IFN-β,IFN-γ, and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-αantibodies, and anti-IFN-γ antibodies), and antibodies thatimmunospecifically bind to tumor-associated antigens (e.g., Herceptin®).In certain embodiments, an immunomodulatory agent is an immunomodulatoryagent other than a chemotherapeutic agent. In other embodiments animmunomodulatory agent is an immunomodulatory agent other than acytokine or hemapoietic such as IL-1, IL-2, IL-4, IL-12, IL-15, TNF,IFN-α, IFN-β, IFN-γ, M-CSF, G-CSF, IL-3 or erythropoietin. In yet otherembodiments, an immunomodulatory agent is an agent other than achemotherapeutic agent and a cytokine or hemapoietic factor.

In a specific embodiment, methods of the invention encompass theadministration of one or more anti-inflammatory agents, such as but notlimited to, non-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

In another specific embodiment, methods of the invention encompass theadministration of one or more antiviral agents (e.g., amantadine,ribavirin, rimantadine, acyclovir, famciclovir, foscarnet, ganciclovir,trifluridine, vidarabine, didanosine, stavudine, zalcitabine,zidovudine, interferon), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)),anti-emetics (e.g., alprazolam, dexamethoasone, domperidone, dronabinol,droperidol, granisetron, haloperidol, haloperidol, iorazepam,methylprednisolone, metoclopramide, nabilone, ondansetron,prochlorperazine), anti-fungal agents (e.g., amphotericin, clotrimazole,econazole, fluconazole, flucytosine, griseofulvin, itraconazole,ketoconazole, miconazole and nystatin), anti-parasite agents (e.g.,dehydroemetine, diloxanide furoate, emetine, mefloquine, melarsoprol,metronidazole, nifurtimox, paromomycin, pentabidine, pentamidineisethionate, primaquine, quinacrine, quinidine) or a combinationthereof.

Specific examples of anti-cancer agents that can be used in variousembodiments of the invention, including pharmaceutical compositions anddosage forms and kits, include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflomithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1;interferon alpha-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (suchas but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin,Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;Vitaxin®; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Additional anti-cancer drugs are 5-fluorouracil andleucovorin. These two agents may be useful when used in methodsemploying thalidomide and a topoisomerase inhibitor. In specificembodiments, an anti-cancer agent is not a chemotherapeutic agent.

In more particular embodiments, the present invention also comprises theadministration of an anti-ICOS mAb in combination with theadministration of one or more therapies such as, but not limited to,anti-cancer agents such as those disclosed in Table 1, for the treatmentof breast, ovary, melanoma, prostate, colon and lung cancers asdescribed above. When used in a combination therapy, the dosages and/orthe frequency of administration listed in Table 1 may be decreased.

TABLE 1 Anti-cancer agents Therapeutic AgentDose/Administration/Formulation doxorubicin Intravenous 60-75 mg/m² onDay 1 21 day intervals hydrochloride (Adriamycin RDF ® and AdriamycinPFS ® epirubicin Intravenous 100-120 mg/m² on Day 1 of 3-4 week cycleshydrochloride each cycle or (Ellence ™) divided equally and given onDays 1-8 of the cycle fluorousacil Intravenous How supplied: 5 mL and 10mL vials (containing 250 and 500 mg flourouracil respectively) docetaxelIntravenous 60-100 mg/m² over 1 hour Once every 3 weeks (Taxotere ®)paclitaxel Intravenous 175 mg/m² over 3 hours Every 3 weeks for(Taxol ®) 4 courses (administered sequentially to doxorubicin-containingcombination chemotherapy) tamoxifen citrate Oral 20-40 mg Daily(Nolvadex ®) (tablet) Dosages greater than 20 mg should be given individed doses (morning and evening) leucovorin intravenous How supplied:Dosage is unclear from calcium for or 350 mg vial text. PDR 3610injection intramuscular injection luprolide acetate single 1 mg (0.2 mLor 20 unit Once a day Lupron ®) subcutaneous mark) injection flutamideOral 50 mg 3 times a day at 8 hour (Eulexin ®) (capsule) (capsulescontain 125 mg intervals (total daily flutamide each) dosage 750 mg)nilutamide Oral 300 mg or 150 mg 300 mg once a day for 30 (Nilandron ®)(tablet) (tablets contain 50 or 150 mg days followed by 150 mgnilutamide each) once a day bicalutamide Oral 50 mg Once a day(Casodex ®) (tablet) (tablets contain 50 mg bicalutamide each)progesterone Injection USP in sesame oil 50 mg/mL ketoconazole Cream 2%cream applied once or (Nizoral ®) twice daily depending on symptomsprednisone Oral Initial dosage may vary from (tablet) 5 mg to 60 mg perday depending on the specific disease entity being treated. estramustineOral 14 mg/kg of body weight Daily given in 3 or 4 phosphate (capsule)(i.e. one 140 mg capsule for divided doses sodium each 10 kg or 22 lb ofbody (Emcyt ®) weight) etoposide or Intravenous 5 mL of 20 mg/mLsolution VP-16 (100 mg) dacarbazine Intravenous 2-4.5 mg/kg Once a dayfor 10 days. (DTIC-Dome ®) May be repeated at 4 week intervalspolifeprosan 20 wafer placed 8 wafers, each containing 7.7 withcarmustine in resection mg of carmustine, for a total implant (BCNU)cavity of 61.6 mg, if size and shape (nitrosourea) of resection cavityallows (Gliadel ®) cisplatin Injection [n/a in PDR 861] How supplied:solution of 1 mg/mL in multi-dose vials of 50 mL and 100 mL mitomycinInjection supplied in 5 mg and 20 mg vials (containing 5 mg and 20 mgmitomycin) gemcitabine HCl Intravenous For NSCLC- 2 schedules 4 weekschedule- (Gemzar ®) have been investigated and Days 1, 8 and 15 of eachthe optimum schedule has 28-day cycle. Cisplatin not been determinedintravenously at 100 4 week schedule- mg/m² on day 1 after theadministration intravenously infusion of Gemzar. at 1000 mg/m² over 30 3week schedule- minutes on 3 week schedule- Days 1 and 8 of each 21Gemzar administered day cycle. Cisplatin at intravenously at 1250 mg/m²dosage of 100 mg/m² over 30 minutes administered intravenously afteradministration of Gemzar on day 1. carboplatin Intravenous Single agenttherapy: Every 4 weeks (Paraplatin ®) 360 mg/m² I.V. on day 1 (infusionlasting 15 minutes or longer) Other dosage calculations: Combinationtherapy with cyclophosphamide, Dose adjustment recommendations, Formuladosing, etc. ifosamide Intravenous 1.2 g/m² daily 5 consecutive days(Ifex ®) Repeat every 3 weeks or after recovery from hematologictoxicity topotecan Intravenous 1.5 mg/m² by intravenous 5 consecutivedays, hydrochloride infusion over 30 minutes starting on day 1 of 21 day(Hycamtin ®) daily course Bisphosphonates Intravenous 60 mg or 90 mgsingle Pamidronate or Oral infusion over 4-24 hours to Alendronate takewith correct hypercalcemia in Risedronate 6-8 oz cancer patients water.5 mg/d daily for 2 years and then 10 mg/d for 9 month to prevent orcontrol bone resorption. 5.0 mg to prevent or control bone resorption.Lovastatin Oral 10-80 mg/day in single or (Mevacor ™) two divided dose.

The invention also encompasses administration of an anti-ICOS mAb incombination with radiation therapy comprising the use of x-rays, gammarays and other sources of radiation to destroy the cancer cells. Inparticular embodiments, the radiation treatment is administered asexternal beam radiation or teletherapy wherein the radiation is directedfrom a remote source. In other embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radiaoactivesource is placed inside the body close to cancer cells or a tumor mass.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (56^(th) ed., 2002).

5.18. Pharmaceutical Compositions

The invention also relates to immunotherapeutic compositions and methodsfor the treatment of T cell-mediated diseases and disorders in humansubjects, such as, but not limited to, chronic infection, autoimmunedisease or disorder, inflammatory disease or disorder, graft-versus-hostdisease (GVHD), transplant rejection, and T cell proliferative disorderin human subjects, using therapeutic antibodies that bind to the ICOSantigen and mediate human ADCC.

The present invention relates to pharmaceutical compositions comprisingeffector function enhanced anti-ICOS antibodies of the IgG1 or IgG3human isotype. The present invention also relates to pharmaceuticalcompositions comprising human or humanized anti-ICOS antibodies of theIgG2 or IgG4 human isotype that mediate human ADCC. In certainembodiments, the present invention also relates to pharmaceuticalcompositions comprising monoclonal anti-ICOS antibodies with enhancedeffectro function that can be produced by means known in the art.

Therapeutic formulations and regimens are described for treating humansubjects diagnosed with autoimmune diseases, such as, but not limitedto, systemic lupus erythematosis, rheumatoid arthritis, immunethrombocytopenic purpura (ITP), diabetes, psoriasis, andhypersensitivity reactions (e.g., allergies, hay fever, asthma, andacute edema cause type I hypersensitivity reactions). The presentinvention also relates to formulations and regimens for the treatment ofhuman subjects diagnosed with chronic inflammatory diseases, such as,but not limited to, inflammatory bowel disease (Crohn's disease andulcerative colitis), Grave's disease, Hashimoto's thyroiditis, anddiabetes mellitus.

Therapeutic formulations and regimens are described for treating humansubjects diagnosed with T cell malignancies that derive from ICOSexpressing T cells and their precursors.

In particular embodiments, anti-ICOS antibodies may mediate ADCC,complement-dependent cellular cytoxicity, or antibody-dependentphagocytosis. Compositions and methods of the present invention alsohave the advantage of targeting a narrower population of T cells thanother T cell directed immunotherapies. For example, anti-ICOS antibodiesof the present invention may be effective to specifically targetactivated T cells, for example, but not limited to, activated T cells.Accordingly, methods and compositions of the invention may be effectiveto reduce or deplete circulating activated CD4+ T cells as well asactivated CD8+ T cells.

Accordingly, in one aspect, the invention provides compositions andmethods for the treatment and prevention of GVHD and graft rejection,which are associated with fewer and/or less severe complications thanless-targeted therapeutic agents and regimens. In one embodiment,compositions and methods of the invention are used with lower doses oftraditional therapeutic agents than would be possible in the absence ofthe methods and compositions of the invention. In another embodiment,compositions and methods of the invention obviate the need for a moresevere form of therapy, such as radiation therapy, high-dosechemotherapy, or splenectomy.

In certain embodiments, anti-ICOS antibodies and compositions may beadministered to a transplant recipient patient prior to or followingtransplantation, alone or in combination with other therapeutic agentsor regimens for the treatment or prevention of GVHD and graft rejection.For example, anti-ICOS antibodies and compositions may be used todeplete activated T cells from a transplant recipient prior to orfollowing transplantation of an allogeneic graft. Anti-ICOS antibodiesand compositions may also be used to deplete activated T cells from thegraft ex vivo, prior to transplantation, or in the donor, or asprophylaxis against GVHD and graft rejection.

5.19. Pharmaceutical Formulations, Administration and Dosing

Pharmaceutical formulations of the invention contain as the activeingredient anti-ICOS antibodies with enhanced effector function. Theformulations contain naked antibody, immunoconjugate, or fusion proteinin an amount effective for producing the desired response in a unit ofweight or volume suitable for administration to a human patient, and arepreferably sterile. The response can, for example, be measured bydetermining the physiological effects of the anti-ICOS antibodycomposition, such as, but not limited to, T cell depletion, IL-17depletion, regression of a T cell malignancy, or decrease of diseasesymptoms. Other assays will be known to one of ordinary skill in the artand can be employed for measuring the level of the response.

5.19.1. Pharmaceutical Formulations

A composition comprising an anti-ICOS antibody with enhanced effectorfunction may be formulated with a pharmaceutically acceptable carrier.The term “pharmaceutically acceptable” means one or more non-toxicmaterials that do not interfere with the effectiveness of the biologicalactivity of the active ingredients. Such preparations may routinelycontain salts, buffering agents, preservatives, compatible carriers, andoptionally other therapeutic agents. Such pharmaceutically acceptablepreparations may also routinely contain compatible solid or liquidfillers, diluents or encapsulating substances which are suitable foradministration into a human. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, boric, formic, malonic, succinic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the antibodies of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy.

According to certain aspects of the invention, anti-ICOS antibodycompositions can be prepared for storage by mixing the antibody orimmunoconjugate having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as histidine, phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

Anti-ICOS antibody compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Anti-ICOS antibody compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, anti-ICOS antibody compositions areprepared by uniformly and intimately bringing the active compound intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of anti-ICOSantibody, which may be isotonic with the blood of the recipient. Thispreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administration canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. In certain embodiments, carrier formulation suitable forvarious routes of administration can be the same or similar to thatdescribed for RITUXAN™. See, Physicians'Desk Reference (MedicalEconomics Company, Inc., Montvale, N.J., 2005), pp. 958-960 and1354-1357, which is incorporated herein by reference in its entirety. Incertain embodiments of the invention, anti-ICOS antibody compositionsare formulated for intravenous administration with sodium chloride,sodium citrate dihydrate, polysorbate 80, and sterile water where the pHof the composition is adjusted to approximately 6.5. Those of skill inthe art are aware that intravenous injection provides a useful mode ofadministration due to the thoroughness of the circulation in rapidlydistributing antibodies. Intravenous administration, however, is subjectto limitation by a vascular barrier comprising endothelial cells of thevasculature and the subendothelial matrix. Still, the vascular barrieris a more notable problem for the uptake of therapeutic antibodies bysolid tumors. Lymphomas have relatively high blood flow rates,contributing to effective antibody delivery. Intralymphatic routes ofadministration, such as subcutaneous or intramuscular injection, or bycatheterization of lymphatic vessels, also provide a useful means oftreating T cell-mediated diseases and disorders. In certain embodiments,anti-ICOS antibodies of compositions and methods of the invention areself-administered subcutaneously. In such embodiments, the compositionis formulated as a lyophilized drug or in a liquid buffer (e.g.,histidine buffer, PBS, citrate) at about 50 mg/mL.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration are typicallysterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing an anti-ICOS antibody, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devized for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions. In certain embodiments, the pharmaceutically acceptablecarriers used in compositions of the invention do not affect human ADCCor CDC.

Anti-ICOS antibody compositions disclosed herein may also be formulatedas immunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug (such as anti-ICOS antibodies disclosed herein) to ahuman. The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.Liposomes containing antibodies of the invention are prepared by methodsknown in the art, such as described in Epstein et al., Proc. Natl. Acad.Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomeswith enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. The antibody of the present invention can be conjugated to theliposomes as described in Martin et al., J. Biol. Chem., 257:286-288(1982) via a disulfide interchange reaction. A therapeutic agent canalso be contained within the liposome. See, Gabizon et al., J. NationalCancer Inst., (19)1484 (1989).

Some of the pharmaceutical formulations include, but are not limited to:

(a) a sterile, preservative-free liquid concentrate for intravenous(i.v.) administration of anti-ICOS antibody, supplied at a concentrationof 10 mg/ml in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.The product can be formulated for i.v. administration using sodiumchloride, sodium citrate dihydrate, polysorbate and sterile water forinjection. For example, the product can be formulated in 9.0 mg/mLsodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mLpolysorbate 80, and sterile water for injection. The pH is adjusted to6.5.

(b) A sterile, lyophilized powder in single-use glass vials forsubcutaneous (s.c.) injection. The product can be formulated withsucrose, L-histidine hydrochloride monohydrate, L-histidine andpolysorbate 20. For example, each single-use vial can contain 150 mganti-ICOS antibody, 123.2 mg sucrose, 6.8 mg L-histidine hydrochloridemonohydrate, 4.3 mg L-histidine, and 3 mg polysorbate 20. Reconstitutionof the single-use vial with 1.3 ml sterile water for injection yieldsapproximately 1.5 ml solution to deliver 125 mg per 1.25 ml (100 mg/ml)of antibody.

(c) A sterile, preservative-free lyophilized powder for intravenous(i.v.) administration. The product can be formulated with α-trehalosedihydrate, L-histidine HCl, histidine and polysorbate 20 USP. Forexample, each vial can contain 440 mg anti-ICOS antibody, 400 mgα,α-trehalose dihydrate, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, and1.8 mg polysorbate 20, USP. Reconstitution with 20 ml of bacteriostaticwater for injection (BWFI), USP, containing 1.1% benzyl alcohol as apreservative, yields a multi-dose solution containing 21 mg/ml antibodyat a pH of approximately 6.

(d) A sterile, lyophilized powder for intravenous infusion in which ananti-ICOS antibody is formulated with sucrose, polysorbate, monobasicsodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.For example, each single-use vial can contain 100 mg antibody, 500 mgsucrose, 0.5 mg polysorbate 80, 2.2 mg monobasic sodium phosphatemonohydrate, and 6.1 mg dibasic sodium phosphate dihydrate. Nopreservatives are present. Following reconstitution with 10 ml sterilewater for injection, USP, the resulting pH is approximately 7.2.

(e) A sterile, preservative-free solution for subcutaneousadministration supplied in a single-use, 1 ml pre-filled syringe. Theproduct can be formulated with sodium chloride, monobasic sodiumphosphate dihydrate, dibasic sodium phosphate dihydrate, sodium citrate,citric acid monohydrate, mannitol, polysorbate 80 and water forinjection, USP. Sodium hydroxide may be added to adjust pH to about 5.2.

For example, each syringe can be formulated to deliver 0.8 ml (40 mg) ofdrug product. Each 0.8 ml contains 40 mg anti-ICOS antibody, 4.93 mgsodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mgdibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 citricacid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80 and water forinjection, USP.

(f) A sterile, preservative-free, lyophilized powder contained in asingle-use vial that is reconstituted with sterile water for injection(SWFI), USP, and administered as a subcutaneous (s.c.) injection. Theproduct can be formulated with sucrose, histidine hydrochloridemonohydrate, L-histidine, and polysorbate. For example, a 75 mg vial cancontain 129.6 mg or 112.5 mg of an anti-ICOS antibody, 93.1 mg sucrose,1.8 mg L-histidine hydrochloride monohydrate, 1.2 mg L-histidine, and0.3 mg polysorbate 20, and is designed to deliver 75 mg of the antibodyin 0.6 ml after reconstitution with 0.9 ml SWFI, USP. A 150 mg vial cancontain 202.5 mg or 175 mg anti-ICOS antibody, 145.5 mg sucrose, 2.8 mgL-histidine hydrochloride monohydrate, 1.8 mg L-histidine, and 0.5 mgpolysorbate 20, and is designed to deliver 150 mg of the antibody in 1.2ml after reconstitution with 1.4 ml SWFI, USP.

(g) A sterile, hyophilized product for reconstitution with sterile waterfor injection. The product can be formulated as single-use vials forintramuscular (IM) injection using mannitol, histidine and glycine. Forexample, each single-use vial can contain 100 mg anti-ICOS antibody,67.5 mg of mannitol, 8.7 mg histidine and 0.3 mg glycine, and isdesigned to deliver 100 mg antibody in 1.0 ml when reconstituted with1.0 ml sterile water for injection. As another example, each single-usevial can contain 50 mg anti-ICOS antibody, 40.5 mg mannitol, 5.2 mghistidine and 0.2 mg glycine, and is designed to deliver 50 mg ofantibody when reconstituted with 0.6 ml sterile water for injection.

(h) A sterile, preservative-free solution for intramuscular (IM)injection, supplied at a concentration of 100 mg/ml. The product can beformulated in single-use vials with histidine, glycine, and sterilewater for injection. For example, each single-use vial can be formulatedwith 100 mg antibody, 4.7 mg histidine, and 0.1 mg glycine in a volumeof 1.2 ml designed to deliver 100 mg of antibody in 1 ml. As anotherexample, each single-use vial can be formulated with 50 mg antibody, 2.7mg histidine and 0.08 mg glycine in a volume of 0.7 ml or 0.5 mldesigned to deliver 50 mg of antibody in 0.5 ml.

In certain embodiments, a pharmaceutical composition of the invention isstable at 4° C. In certain embodiments, a pharmaceutical composition ofthe invention is stable at room temperature.

In one embodiment, a liquid formulation of the invention is an aqueousformulation. In a specific embodiment, a liquid formulation of theinvention is an aqueous formulation wherein the aqueous carrier isdistilled water.

In one embodiment, a formulation of the invention is sterile. In oneembodiment, a formulation of the invention is homogeneous. In oneembodiment, a formulation of the invention is isotonic.

In one embodiment, a formulation of the invention comprises at leastabout 1 mg/ml, at least about 5 mg/ml, at least about 10 mg/ml, at leastabout 20 mg/ml, at least about 30 mg/ml, at least about 40 mg/ml, atleast about 50 mg/ml, at least about 60 mg/ml, at least about 70 mg/ml,at least about 80 mg/ml, at least about 90 mg/ml, at least about 100mg/ml, at least about 110 mg/ml, at least about 120 mg/ml, at leastabout 130 mg/ml, at least about 140 mg/ml, at least about 150 mg/ml, atleast about 160 mg/ml, at least about 170 mg/ml, at least about 180mg/ml, at least about 190 mg/ml, at least about 200 mg/ml, or at leastabout 300 mg/ml of an anti-ICOS antibody or a fragment thereof.

Optionally, the formulations of the invention may comprise commonexcipients and/or additives such as buffering agents, saccharides, saltsand surfactants. Additionally or alternatively, the formulations of theinvention may further comprise common excipients and/or additives, suchas, but not limited to, solubilizers, diluents, binders, stabilizers,salts, lipophilic solvents, amino acids, chelators, preservatives, orthe like.

In certain embodiments, the buffering agent is selected from the groupconsisting of histidine, citrate, phosphate, glycine, and acetate. Inother embodiments the saccharide excipient is selected from the groupconsisting of trehalose, sucrose, mannitol, maltose and raffinose. Instill other embodiments the surfactant is selected from the groupconsisting of polysorbate 20, polysorbate 40, polysorbate 80, andPluronic F68. In yet other embodiments the salt is selected from thegroup consisting of NaCl, KCl, MgCl₂, and CaCl₂

Optionally, the formulations of the invention may further comprise othercommon auxiliary components, such as, but not limited to, suitableexcipients, polyols, solubilizers, diluents, binders, stabilizers,lipophilic solvents, chelators, preservatives, or the like.

The formulations of the invention include a buffering or pH adjustingagent to provide improved pH control. In one embodiment, a formulationof the invention has a pH of between about 3.0 and about 9.0, betweenabout 4.0 and about 8.0, between about 5.0 and about 8.0, between about5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5and about 8.0, between about 5.5 and about 7.0, or between about 5.5 andabout 6.5. In a further embodiment, a formulation of the invention has apH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, aformulation of the invention has a pH of about 6.0.

The pH of the formulation generally should not be equal to theisoelectric point of the particular antibody (including antibodyfragment thereof) to be used in the formulation (for example, but notlimited to, the isoelectric point of 13H5, 13H7 or 7H9) and may rangefrom about 4.0 to about 8.0, or may range from about 5.5 to about 6.5.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is selected from the groupconsisting of histidine, citrate, phosphate, glycine, and acetate. In aspecific embodiment, the buffering agent is histidine. In anotherspecific embodiment, the buffering agent is citrate. The purity of thebuffering agent should be at least 98%, or at least 99%, or at least99.5%. As used herein, the term “purity” in the context of histidinerefers to chemical purity of histidine as understood in the art, e.g.,as described in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In certain embodiments, aformulation of the invention comprises a buffering agent. In oneembodiment, said buffering agent is selected from the group consistingof histidine, citrate, phosphate, glycine, and acetate. In a specificembodiment, a formulation of the invention comprises histidine as abuffering agent. In a further embodiment, a formulation of the inventioncomprises a citrate buffer.

In one embodiment, a formulation of the invention comprises at leastabout 1 mM, at least about 5 mM, at least about 10 mM, at least about 20mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, atleast about 75 mM, at least about 100 mM, at least about 150 mM, or atleast about 200 mM buffering agent.

In certain embodiments, the formulations of the invention comprise acarbohydrate excipient. Carbohydrate excipients can act, e.g., asviscosity enhancing agents, stabilizers, bulking agents, solubilizingagents, and/or the like. Carbohydrate excipients are generally presentat between about 1% to about 99% by weight or volume. In one embodiment,the carbohydrate excipient is present at between about 0.1% to about20%. In another embodiment, the carbohydrate excipient is present atbetween about 0.1% to about 15%. In a specific embodiment, thecarbohydrate excipient is present at between about 0.1% to about 5%, orbetween about 1% to about 20%, or between about 5% to about 15%, orbetween about 8% to about 10%, or between about 10% and about 15%, orbetween about 15% and about 20%. In another specific embodiment, thecarbohydrate excipient is present at between 0.1% to 20%, or between 5%to 15%, or between 8% to 10%, or between 10% and 15%, or between 15% and20%. In still another specific embodiment, the carbohydrate excipient ispresent at between about 0.1% to about 5%. In still another specificembodiment, the carbohydrate excipient is present at between about 5% toabout 10%. In yet another specific embodiment, the carbohydrateexcipient is present at between about 15% to about 20%. In still otherspecific embodiments, the carbohydrate excipient is present at 1%, or at1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, orat 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, for example, monosaccharides such as fructose,maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are selected from the group consisting of, sucrose, trehalose,lactose, mannitol, and raffinose. In a specific embodiment, thecarbohydrate excipient is trehalose. In another specific embodiment, thecarbohydrate excipient is mannitol. In yet another specific embodiment,the carbohydrate excipient is sucrose. In still another specificembodiment, the carbohydrate excipient is raffinose. The purity of thecarbohydrate excipient should be at least 98%, or at least 99%, or atleast 99.5%.

In one embodiment, a formulation of the invention comprises anexcipient. In a specific embodiment, a formulation of the inventioncomprises at least one excipient selected from the group consisting of:sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifierand preservative. In one embodiment, a formulation of the inventioncomprises a salt. In one embodiment, a formulation of the inventioncomprises a salt selected from the group consisting of: NaCl, KCl,CaCl₂, and MgCl₂. In a specific embodiment, a formulation of theinvention comprises NaCl.

In one embodiment, a formulation of the invention comprises at leastabout 10 mM, at least about 25 mM, at least about 50 mM, at least about75 mM, at least about 100 mM, at least about 125 mM, at least about 150mM, at least about 175 mM. at least about 200 mM, or at least about 300mM sodium chloride.

The formulations of the invention may further comprise a surfactant. Theterm “surfactant” as used herein refers to organic substances havingamphipathic structures; namely, they are composed of groups of opposingsolubility tendencies, typically an oil-soluble hydrocarbon chain and awater-soluble ionic group. Surfactants can be classified, depending onthe charge of the surface-active moiety, into anionic, cationic, andnonionic surfactants. Surfactants are often used as wetting,emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.polysorbates 20 or 80); polyoxamers (e.g. poloxamer 188); Triton; sodiumoctyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine;lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-,palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl-, or disodium methyl oleyl-taurate; and the MONAQUA™ series (MonaIndustries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol,and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68etc), can optionally be added to the formulations of the invention toreduce aggregation. Surfactants are particularly useful if a pump orplastic container is used to administer the formulation. The presence ofa pharmaceutically acceptable surfactant mitigates the propensity forthe protein to aggregate. In a specific embodiment, the formulations ofthe invention comprise a polysorbate which is at a concentration rangingfrom between about 0.001% to about 1%, or about 0.001% to about 0.1%, orabout 0.01% to about 0.1%. In other specific embodiments, theformulations of the invention comprise a polysorbate which is at aconcentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.In another specific embodiment, the polysorbate is polysorbate-80.

In one embodiment, a formulation of the invention comprises asurfactant. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80. In aspecific embodiment, a formulation of the invention comprisesPolysorbate 80.

Optionally, the formulations of the invention may further comprise othercommon excipients and/or additives including, but not limited to,diluents, binders, stabilizers, lipophilic solvents, preservatives,adjuvants, or the like. Pharmaceutically acceptable excipients and/oradditives may be used in the formulations of the invention. Commonlyused excipients/additives, such as pharmaceutically acceptable chelators(for example, but not limited to, EDTA, DTPA or EGTA) can optionally beadded to the formulations of the invention to reduce aggregation. Theseadditives are particularly useful if a pump or plastic container is usedto administer the formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield an microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA)), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofFc variant protein as well known in the art or as described herein.

It will be understood by one skilled in the art that the formulations ofthe invention may be isotonic with human blood, that is the formulationsof the invention have essentially the same osmotic pressure as humanblood. Such isotonic formulations will generally have an osmoticpressure from about 250 mOSm to about 350 mOSm. Isotonicity can bemeasured by, for example, using a vapor pressure or ice-freezing typeosmometer. Tonicity of a formulation is adjusted by the use of tonicitymodifiers. “Tonicity modifiers” are those pharmaceutically acceptableinert substances that can be added to the formulation to provide anisotonity of the formulation. Tonicity modifiers suitable for thisinvention include, but are not limited to, saccharides, salts and aminoacids.

In certain embodiments, the formulations of the present invention havean osmotic pressure from about 100 mOSm to about 1200 mOSm, or fromabout 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSmto about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or fromabout 250 mOSm to about 350 mOSm.

Concentration of any one or any combination of various components of theformulations of the invention are adjusted to achieve the desiredtonicity of the final formulation. For example, the ratio of thecarbohydrate excipient to antibody may be adjusted according to methodsknown in the art (e.g., U.S. Pat. No. 6,685,940). In certainembodiments, the molar ratio of the carbohydrate excipient to antibodymay be from about 100 moles to about 1000 moles of carbohydrateexcipient to about 1 mole of antibody, or from about 200 moles to about6000 moles of carbohydrate excipient to about 1 mole of antibody, orfrom about 100 moles to about 510 moles of carbohydrate excipient toabout 1 mole of antibody, or from about 100 moles to about 600 moles ofcarbohydrate excipient to about 1 mole of antibody.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Salts that arepharmaceutically acceptable and suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the inventions comprise NaCl, MgCl₂, and/or CaCl₂. Inone embodiment, concentration of NaCl is between about 75 mM and about150 mM. In another embodiment, concentration of MgCl₂ is between about 1mM and about 100 mM. Amino acids that are pharmaceutically acceptableand suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the invention are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in amounts of several hundred orthousand milligrams per kilogram body weight, as can be the case withantibodies, even trace amounts of harmful and dangerous endotoxin mustbe removed. In certain specific embodiments, the endotoxin and pyrogenlevels in the composition are less then 10 EU/mg, or less then 5 EU/mg,or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg,or less then 0.001 EU/mg.

When used for in vivo administration, the formulations of the inventionshould be sterile. The formulations of the invention may be sterilizedby various sterilization methods, including sterile filtration,radiation, etc. In one embodiment, the antibody formulation isfilter-sterilized with a presterilized 0.22-micron filter. Sterilecompositions for injection can be formulated according to conventionalpharmaceutical practice as described in “Remington: The Science &Practice of Pharmacy”, 21^(st) ed., Lippincott Williams & Wilkins,(2005). Formulations comprising antibodies, such as those disclosedherein, ordinarily will be stored in lyophilized form or in solution. Itis contemplated that sterile compositions comprising antibodies areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having an adapter that allows retrievalof the formulation, such as a stopper pierceable by a hypodermicinjection needle.

The terms “stability” and “stable” as used herein in the context of aformulation comprising an anti-ICOS antibody of the invention refer tothe resistance of the antibody in the formulation to aggregation,degradation or fragmentation under given manufacture, preparation,transportation and storage conditions. The “stable” formulations of theinvention retain biological activity under given manufacture,preparation, transportation and storage conditions. The stability ofsaid antibody can be assessed by degrees of aggregation, degradation orfragmentation, as measured by HPSEC, static light scattering (SLS),Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD),urea unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS binding techniques,compared to a reference formulation. For example, a referenceformulation may be a reference standard frozen at −70° C. consisting of10 mg/ml of an anti-ICOS antibody of the invention in PBS. The overallstability of a formulation comprising an anti-ICOS antibody of theinvention can be assessed by various assays including, for example,ELISA assay, radioimmunoassay and ADCC assay. The overall stability of aformulation comprising an anti-ICOS antibody of the invention can alsobe assessed by in vivo assays including, for example, in vivo depletionassays.

In one embodiment, a formulation of the invention comprises an anti-ICOSantibody. In one embodiment, a formulation of the invention reducesaggregation of an anti-ICOS antibody or fragment thereof. In anotherembodiment, a formulation of the invention reduces fragmentation of ananti-ICOS antibody or fragment thereof. In a further embodiment, aformulation of the invention reduces deamidation of an anti-ICOSantibody or fragment thereof.

In one embodiment, a formulation of the invention comprises an anti-ICOSantibody of the invention and is stable upon storage at about 40° C. forat least about 1 week, at least about 2 weeks, at least about 3 weeks,or at least about 4 weeks. In one embodiment, a formulation of theinvention is stable upon storage at about 40° C. for at least about 1month, at least about 2 months, at least about 3 months, at least about4 months, at least about 5 months, or at least about 6 months.

In one embodiment, a formulation of the invention comprises an anti-ICOSantibody of the invention and is stable upon storage at about 5° C. forat least about 1 month, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 10 months, at least about 11 months, orat least about 12 months. In one embodiment, a formulation of theinvention is stable upon storage at about 5° C. for at least about 1year, at least about 2 years, at least about 3 years, at least about 4years, at least about 5 years, at least about 6 years, at least about 7years, at least about 8 years, at least about 9 years, at least about 10years, at least about 11 years, or at least about 12 years.

In a specific embodiment, a formulation of the invention comprises atleast about 50 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 40° C. for at leastabout 1 week, at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 2 months, at least about 3 months, atleast about 4 months, at least about 5 months, or at least about 6months.

In a specific embodiment, a formulation of the invention comprises atleast about 50 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 5C for at least about 6months, at least about 7 months, at least about 8 months, at least about9 months, at least about 1 year, at least about 2 years, or at leastabout 3 years.

In a specific embodiment, a formulation of the invention comprises atleast about 100 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 40° C. for at leastabout 1 week, at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 2 months, at least about 3 months, atleast about 4 months, at least about 5 months, or at least about 6months.

In a specific embodiment, a formulation of the invention comprises atleast about 100 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 5° C. for at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 1 year, at least about 2 years, or atleast about 3 years.

In a specific embodiment, a formulation of the invention comprises atleast about 110 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 40° C. for at leastabout 1 week, at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 2 months, at least about 3 months, atleast about 4 months, at least about 5 months, or at least about 6months.

In a specific embodiment, a formulation of the invention comprises atleast about 110 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 5° C. for at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 1 year, at least about 2 years, or atleast about 3 years.

In a specific embodiment, a formulation of the invention comprises atleast about 150 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 40° C. for at leastabout 1 week, at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 2 months, at least about 3 months, atleast about 4 months, at least about 5 months, or at least about 6months.

In a specific embodiment, a formulation of the invention comprises atleast about 150 mg/ml of an anti-ICOS antibody described herein, whereinthe formulation is stable upon storage at about 5° C. for at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 1 year, at least about 2 years, or atleast about 3 years.

5.19.2. Antibody Half-Life

In certain embodiments, the half-life of an anti-ICOS antibody ofcompositions and methods of the invention is at least about 4 to 7 days.In certain embodiments, the mean half-life of an anti-ICOS antibody ofcompositions and methods of the invention is at least about 2 to 5 days,3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to11 days, 8 to 12, 9 to 13, 10 to 14, 11 to 15, 12 to 16, 13 to 17, 14 to18, 15 to 19, or 16 to 20 days. In other embodiments, the mean half-lifeof an anti-ICOS antibody of compositions and methods of the invention isat least about 17 to 21 days, 18 to 22 days, 19 to 23 days, 20 to 24days, 21 to 25, days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to29 days, or 26 to 30 days. In still further embodiments the half-life ofan anti-ICOS antibody of compositions and methods of the invention canbe up to about 50 days. In certain embodiments, the half-lives ofantibodies of compositions and methods of the invention can be prolongedby methods known in the art. Such prolongation can in turn reduce theamount and/or frequency of dosing of the antibody compositions.Antibodies with improved in vivo half-lives and methods for preparingthem are disclosed in U.S. Pat. No. 6,277,375; and InternationalPublication Nos. WO 98/23289 and WO 97/3461.

The serum circulation of anti-ICOS antibodies in vivo may also beprolonged by attaching inert polymer molecules such as high molecularweight polyethyleneglycol (PEG) to the antibodies with or without amultifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of the antibodies or via epsilon-aminogroups present on lysyl residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure proper conjugation of PEG molecules tothe antibodies. Unreacted PEG can be separated from antibody-PEGconjugates by size-exclusion or by ion-exchange chromatography.PEG-derivatized antibodies can be tested for binding activity as well asfor in vivo efficacy using methods known to those of skill in the art,for example, by immunoassays described herein.

Further, the antibodies of compositions and methods of the invention canbe conjugated to albumin in order to make the antibody more stable invivo or have a longer half-life in vivo. The techniques are well knownin the art, see, e.g., International Publication Nos. WO 93/15199, WO93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all ofwhich are incorporated herein by reference.

Additionally, variant Fc regions that confer increased in vivo half-lifeon antibodies has been described (see, US Patent Publication No:US2003/0190311 A1). The use of Fc variants with extended in vivohalf-life in combination with the compositions and methods of thecurrent invention is contemplated. In one embodiment, an anti-ICOSantibody of the invention comprises a variant Fc region with increasedin vivo half-life. In a further embodiment, an anti-ICOS antibody of theinvention comprises a varian tfc region comprising at least onesubstitution of an amino acid residue selected from the group consistingof: residue 252, 254, and 256, wherein the amino acid residue positionsare determined according to the EU convention. In a specific embodiment,an anti-ICOS antibody of the invention comprises a variant Fc regioncomprising at least one amino acid substitution selected from the groupconsisting of: M252Y, S254T, and T256E; wherein the amino acid residuepositions are determined according to the EU convention. In a furtherembodiment, an anti-ICOS antibody of the invention comprises a variantFc region comprising at least one amino acid residue selected from thegroup consisting of: Y at position 252, T at position 254, and E atposition 256; wherein the amino acid residue positions are determinedaccording to the EU convention.

5.19.3. Administration and Dosing

Administration of compositions of the invention to a human patient canbe by any route, including but not limited to intravenous, intradermal,transdermal, subcutaneous, intramuscular, inhalation (e.g., via anaerosol), buccal (e.g., sub-lingual), topical (i.e., both skin andmucosal surfaces, including airway surfaces), intrathecal,intraarticular, intraplural, intracerebral, intra-arterial,intraperitoneal, oral, intralymphatic, intranasal, rectal or vaginaladministration, by perfusion through a regional catheter, or by directintralesional injection. In one embodiment, compositions of theinvention are administered by intravenous push or intravenous infusiongiven over defined period (e.g., 0.5 to 2 hours). Compositions of theinvention can be delivered by peristaltic means or in the form of adepot, although the most suitable route in any given case will depend,as is well known in the art, on such factors as the species, age, genderand overall condition of the subject, the nature and severity of thecondition being treated and/or on the nature of the particularcomposition (i.e., dosage, formulation) that is being administered. Inparticular embodiments, the route of administration is via bolus orcontinuous infusion over a period of time, once or twice a week. Inother particular embodiments, the route of administration is bysubcutaneous injection, optionally once or twice weekly. In oneembodiment, compositions, and/or methods of the invention areadministered on an outpatient basis.

In certain embodiments, the dose of a composition comprising anti-ICOSantibody is measured in units of mg/kg of patient body weight. In otherembodiments, the dose of a composition comprising anti-ICOS antibody ismeasured in units of mg/kg of patient lean body weight (i.e., bodyweight minus body fat content). In yet other embodiments, the dose of acomposition comprising anti-ICOS antibody is measured in units of mg/m²of patient body surface area. In yet other embodiments, the dose of acomposition comprising anti-ICOS antibody is measured in units of mg perdose administered to a patient. Any measurement of dose can be used inconjunction with compositions and methods of the invention and dosageunits can be converted by means standard in the art.

Those skilled in the art will appreciate that dosages can be selectedbased on a number of factors including the age, sex, species andcondition of the subject (e.g., stage of disease), the desired degree ofcellular depletion, the disease to be treated and/or the particularantibody or antigen-binding fragment being used and can be determined byone of skill in the art. For example, effective amounts of compositionsof the invention may be extrapolated from dose-response curves derivedin vitro test systems or from animal model (e.g., the cotton rat ormonkey) test systems. Models and methods for evaluation of the effectsof antibodies are known in the art (Wooldridge et al., Blood, 89(8):2994-2998 (1997)), incorporated by reference herein in its entirety). Incertain embodiments, for particular ICOS expressing T cell malignancies,therapeutic regimens standard in the art for antibody therapy can beused with compositions and methods of the invention.

Examples of dosing regimens that can be used in methods of the inventioninclude, but are not limited to, daily, three times weekly(intermittent), weekly, or every 14 days. In certain embodiments, dosingregimens include, but are not limited to, monthly dosing or dosing every6-8 weeks.

Those skilled in the art will appreciate that dosages are generallyhigher and/or frequency of administration greater for initial treatmentas compared with maintenance regimens.

In some embodiments of the invention, anti-ICOS antibodies bind to ICOSexpressing T cells and may result in efficient (i.e., at low dosage)depletion of ICOS expressing T cells (as described herein). In certainembodiments, dosages of the antibody (optionally in a pharmaceuticallyacceptable carrier as part of a pharmaceutical composition) are at leastabout 0.0005, 0.001, 0.05, 0.075, 0.1, 0.25, 0.375, 0.5, 1, 2.5, 5, 10,20, 37.5, or 50 mg/m² and/or less than about 500, 475, 450, 425, 400,375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 60, 50,37.5, 20, 15, 10, 5, 2.5, 1, 0.5, 0.375, 0.1, 0.075 or 0.01 mg/m². Incertain embodiments, the dosage is between about 0.0005 to about 200mg/m², between about 0.001 and 150 mg/m², between about 0.075 and 125mg/m², between about 0.375 and 100 mg/m², between about 2.5 and 75mg/m², between about 10 and 75 mg/m², and between about 20 and 50 mg/m².In related embodiments, the dosage of anti-ICOS antibody used is atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of a patient. In certainembodiments, the dose of naked anti-ICOS antibody used is at least about1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg of body weight of apatient. In certain embodiments, the dose of anti-ICOS antibody used isat least about 1 to 20, 3 to 15, or 5 to 10 mg/kg of body weight of apatient. In other embodiments, the dose of anti-ICOS antibody used is atleast about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of a patient. Incertain embodiments, a single dosage unit of the antibody (optionally ina pharmaceutically acceptable carrier as part of a pharmaceuticalcomposition) can be at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, or 250 micrograms/m². In otherembodiments, dose is up to 1 g per single dosage unit.

In some embodiments of methods of this invention, antibodies and/orcompositions of this invention can be administered at a dose lower thanabout 375 mg/m²; at a dose lower than about 37.5 mg/m²; at a dose lowerthan about 0.375 mg/m²; and/or at a dose between about 0.075 mg/m² andabout 125 mg/m². In certain embodiments of methods of the invention,dosage regimens comprise low doses, administered at repeated intervals.For example, in one embodiment, compositions of the invention can beadministered at a dose lower than about 375 mg/m² at intervals ofapproximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 days.

The specified dosage can result in ICOS expressing T cell depletion inthe human treated using compositions and methods of the invention for aperiod of at least about 1, 2, 3, 5, 7, 10, 14, 20, 30, 45, 60, 75, 90,120, 150 or 180 days or longer. In certain embodiments of methods of theinvention, ICOS expressing T cells are depleted by at least 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% in comparison to ICOS expressing T celllevels in the patient being treated before use of compositions andmethods of the invention. In other embodiments of methods of theinvention, ICOS expressing T cells are depleted by at least 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% in comparison to typical standard ICOSexpressing T cell levels for humans. In related embodiments, the typicalstandard ICOS expressing T cell levels for humans are determined usingpatients comparable to the patient being treated with respect to age,sex, weight, and other factors.

In certain embodiments of the invention, a dosage of about 125 mg/m² orless of an antibody or antigen-binding fragment results in ICOSexpressing T cell depletion for a period of at least about 7, 14, 21,30, 45, 60, 90, 120, 150, or 200 days. In another representativeembodiment, a dosage of about 37.5 mg/m² or less depletes ICOSexpressing T cells for a period of at least about 7, 14, 21, 30, 45, 60,90, 120, 150, or 200 days. In still other embodiments, a dosage of about0.375 mg/m² or less results in depletion of ICOS expressing T cells forat least about 7, 14, 21, 30, 45 or 60 days. In another embodiment, adosage of about 0.075 mg/m² or less results in depletion of ICOSexpressing T cells for a period of at least about 7, 14, 21, 30, 45, 60,90, 120, 150, or 200 days. In yet other embodiments, a dosage of about0.01 mg/m², 0.005 mg/m² or even 0.001 mg/m² or less results in depletionof ICOS expressing T cells for at least about 3, 5, 7, 10, 14, 21, 30,45, 60, 90, 120, 150, or 200 days. According to these embodiments, thedosage can be administered by any suitable route, but is optionallyadministered by a subcutaneous route.

As another aspect, the invention provides the discovery that ICOSexpressing T cell depletion and/or treatment of T cell-mediateddisorders can be achieved at lower dosages of antibody or antibodyfragments than employed in currently available methods. Thus, in anotherembodiment, the invention provides a method of depleting ICOS expressingT cells and/or treating a T cell-mediated disorder, comprisingadministering to a human, an effective amount of an antibody thatspecifically binds to ICOS, wherein a dosage of about 500, 475, 450,425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100,75, 60, 50, 37.5, 20, 10, 5, 2.5, 1, 0.5, 0.375, 0.25, 0.1, 0.075, 0.05,0.001, 0.0005 mg/m² or less results in a depletion of ICOS expressing Tcells (circulating and/or tissue ICOS expressing T cells) of 25%, 35%,50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or more for a period at leastabout 3, 5, 7, 10, 14, 21, 30, 45, 60, 75, 90, 120, 150, 180, or 200days or longer. In representative embodiments, a dosage of about 125mg/m² or 75 mg/m² or less results in at least about 50%, 75%, 85% or 90%depletion of ICOS expressing T cells for at least about 7, 14, 21, 30,60, 75, 90, 120, 150 or 180 days. In other embodiments, a dosage ofabout 50, 37.5 or 10 mg/m² results in at least about a 50%, 75%, 85% or90% depletion of ICOS expressing T cells for at least about 7, 14, 21,30, 60, 75, 90, 120 or 180 days. In still other embodiments, a dosage ofabout 0.375 or 0.1 mg/m² results in at least about a 50%, 75%, 85% or90% depletion of ICOS expressing T cells for at least about 7, 14, 21,30, 60, 75 or 90 days. In further embodiments, a dosage of about 0.075,0.01, 0.001, or 0.0005 mg/m² results in at least about a 50%, 75%, 85%or 90% depletion of ICOS expressing T cells for at least about 7, 14,21, 30 or 60 days.

In certain embodiments of the invention, the dose can be escalated orreduced to maintain a constant dose in the blood or in a tissue, suchas, but not limited to, bone marrow. In related embodiments, the dose isescalated or reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, and 95% in order to maintain a desired level of anantibody of compositions and methods of the invention.

In certain embodiments, the dosage can be adjusted and/or the infusionrate can be reduced based on patient's immunogenic response tocompositions and methods of the invention.

5.19.4. Toxicity Testing

The tolerance, toxicity and/or efficacy of the compositions and/ortreatment regimens of the present invention can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation), the ED50 (the dose therapeutically effective in 50% of thepopulation), and IC50 (the dose effective to achieve a 50% inhibition).In one embodiment, the dose is a dose effective to achieve at least a60%, 70%, 80%, 90%, 95%, or 99% depletion of circulating ICOS expressingT cells. The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD50/ED50.Therapies that exhibit large therapeutic indices may be preferred. Whiletherapies that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such agents toICOS-expressing cells in order to minimize potential damage to ICOSnegative cells and, thereby, reduce side effects.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages of the compositions and/ortreatment regimens for use in humans. The dosage of such agents may liewithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any therapy used in methods of the invention, a therapeuticallyeffective dose can be estimated by appropriate animal models. Dependingon the species of the animal model, the dose can be scaled for human useaccording to art-accepted formulas, for example, as provided byFreireich et al., Quantitative comparison of toxicity of anticanceragents in mouse, rat, monkey, dog, and human, Cancer ChemotherapyReports, NCI 1966 40:219-244. Data obtained from cell culture assays canbe useful for predicting potential toxicity. Animal studies can be usedto formulate a specific dose to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Plasma drug levels may bemeasured, for example, by high performance liquid chromatography, ELISA,or by cell based assays.

5.20. Therapeutic Uses

Compositions comprising an anti-ICOS antibody with enhanced effectorfunction may be used for the treatment of autoimmune diseases, such assystemic lupus erythematosis, rheumatoid arthritis, multiple sclerosis,diabetes, immune thrombocytopenic purpura (ITP), and psoriasis; chronicinflammatory diseases, such as inflammatory bowel disease (Crohn'sdisease and ulcerative colitis), Grave's disease, Hashimoto'sthyroiditis, and diabetes mellitis. Anti-ICOS compositions describedherein may also be used to alleviate toxic shock syndrome, inflammatorybowel disease, allosensitization due to blood transfusions, T-celldependent B-cell-mediated diseases, and the treatment of graft vs. hostdisease. In addition, compositions and methods of the invention may beuseful in therapeutic indications that call for the inhibition orenhancement of antibody production.

Compositions comprising an anti-ICOS antibody with enhanced effectorfunction may also be used as immunosuppressive agents for bone marrowand organ transplantation and may be used to prolong graft survival.Such compositions may provide significant advantages over existingtreatment. Bone marrow and organ transplantation therapy must contendwith T-cell-mediated rejection of the foreign cells or tissue by thehost. Present therapeutic regimens for inhibiting T-cell-mediatedrejection involve treatment with the drugs cyclosporine or FK506. Whiledrugs are effective, patients suffer from serious side effects,including hepatotoxicity, nephrotoxicity, and neurotoxicity. The targetfor the cyclosporin/FK506 class of therapeutics is calcineurin, aphosphatase with ubiquitous expression. Since ICOS expression isrestricted to T-cells, depletion of ICOS expressing T cells may lack thesevere side effects observed with the use of the presentimmunotherapeutic agents.

Hypersensitivity is a normally beneficial immune response that isexaggerated or inappropriate, and leads to inflammatory reactions andtissue damage. Hypersensitivity reactions which are antibody-mediatedmay be particularly susceptible to antagonism by depletion of ICOSexpressing cells. Allergies, hay fever, asthma, and acute edema causetype I hypersensitivity reactions, and these reactions may be suppressedby depletion of ICOS expressing cells.

Diseases that cause antibody-mediated hypersensitivity reactions,including systemic lupus erythematosis, arthritis (rheumatoid arthritis,reactive arthritis, psoriatic arthritis), nephropathies(glomerulo-nephritis, membranous, mesangiocapillary, focal segmental,focal necrotizing, crescentic, proliferative—tubulopathies), skindisorders (pemphigus and pemphigoid, erythema nodosum), endocrinopathies(thyroiditis—Grave's, Hashimoto's—insulin dependent diabetes mellitus),various pneumopathies (especially extrinsic alveolitis), variousvasculopathies, coeliac disease, with aberrant production of IgA, manyanemias and thrombocytopenias, Guillain-Barre Syndrome, and myastheniagravis, may be treated using compositions comprising an anti-ICOSantibody with enhanced effector function.

In addition, lymphoproliferative disorders, such as multiple myeloma,Waldenstrom's macroglobulinemia, and crioglobulinemias may be inhibitedby administering a composition comprising an anti-ICOS antibody withenhanced effector function. Additionally, graft versus host disease, an“artificial” immune disorder, may benefit from the depletion of ICOSexpressing cells.

The ICOS dependent co-stimulatory pathway is involved in regulating IgEproduction. IgE is an immunoglobulin isotype specifically involved inmediating allergic responses such as asthma, food allergies, hay fever,type 1 hypersensitivity and sinus inflammation. Upon exposure to anallergen, a process involving T-cell and B cell collaboration results inB cell production of IgE specific for the allergen. Allergen-specificIgE released into the circulation by B cells bind to mast cells andbasophils through the high affinity IgE receptor (FceRI). Mast cells andbasophils to which IgE is bound become sensitized and subsequentexposure to the allergen results in cross-linking of the surfacereceptors and release of histamines.

The invention provides for the use of an anti-ICOS antibody to regulateIgE production and to prevent or treat IgE-mediated disorders. By way ofexample, such disorders include allergic responses such as asthma, foodallergies, hay fever, hypersensitivity, and sinus inflammation. In oneembodiment, an anti-ICOS antibody of the invention is used to partiallyor completely inhibit IgE production. An anti-ICOS antibody of theinvention may be used separately, or in combination, in a treatmentregimen for decreasing IgE levels.

The invention also provides for the use of an anti-ICOS antibody incombination with an IgE antagonist to partially or completely inhibitIgE production and to prevent and/or treat disorders characterized byexcessive or inappropriate IgE production. As used herein the term “IgEantagonist” refers to a compound capable of disrupting or blocking theinteraction of IgE with its high affinity receptor FceRI on cells suchthat the response to allergen stimulus is attenuated or eliminated.Antagonists include an anti-IgE antibody and fragments thereof, solubleFceRI receptor and fragments thereof, anti-FceRI antibody and fragmentsthereof, IgE variants and fragments thereof, IgE binding peptides, FceRIreceptor binding peptides, and small molecules capable of binding to IgEor competing with IgE for binding to FceRI receptor. An anti-ICOSantibody of the invention may also be used with in combination withantihistamines, allergen desensitization, reduction in exposure toallergen and the like for treatment of allergic disorders.

The invention also provides for the prevention and/or treatment ofasthma comprising administering an anti-ICOS antibody of the inventionalone or in conjunction with one or more agents for treating asthma.Examples of such agents include bronchodilators (anti-cholinergicagents, .beta-2 adrenergic receptor agonists, lenkotriene D-4antagonists, neurokinin antagonists, potassium channel openers,substance P antagonists, thromboxane A-2 antagonists, and xanthines),anti-inflammatories (5-lipoxygenase inhibitors, 5-lipoxygenaseactivating protein inhibitors, phosphodiesterase IV inhibitors, plateletactivating factor antagonists, respiratory NSAIDS, steroids, andtyrosine kinase inhibitors), cytokine inhibitors (CD4, IL-4 and IL-5inhibitors) and IgE antagonists as set forth above.

Compositions and methods according to this invention are able to control(suppress or stimulate) proliferation of ICOS expressing cells orproduction of cytokine (for example, IL-17) by ICOS expressing cells,thereby enabling suppression of various pathological conditions andtreatment or prevention of various disorders caused by diversephysiological phenomena related to signal transduction mediated by ICOS.

Compositions comprising an anti-ICOS antibody of this invention enablessuppression, prevention and/or treatment of, for example, but notlimited to, rheumatoid arthritis, multiple sclerosis, autoimmunethyroiditis, allergic contact-type dermatitis, chronic inflammatorydermatosis (e.g., lichen planus), systemic lupus erythematosus,insulin-dependent diabetes mellitus, psoriasis, autoimmune or allergicdisorders, autoimmune disease and delayed allergy caused by cellularimmunity; arthropathia (for example, but not limited to, rheumatoidarthritis (RA) and osteoarthritis (OA)), inflammation (e.g., hepatitis),graft versus host reaction (GVH reaction), graft versus host disease(GVHD), immune rejection accompanying transplantation of a tissue (e.g.,skin, cornea, bone) or organ (e.g., liver, heart, lung, kidney,pancreas), immune response triggered by a foreign antigen or autoantigen(for example, production of antibodies against said antigen, cellproliferation, production of cytokines), and disorders caused by theabnormal intestinal immunity (e.g., inflammatory intestinal disorders,Crohn's disease, ulcerative colitis, alimentary allergy).

Furthermore, compositions and methods described herein may be utilizedfor the suppression/treatment of transplant rejection or GVHD incombination with known immunosuppressive agents such as inhibitors ofcytokine transcription (e.g., cyclosporin A, tacrolimus), nucleotidesynthesis (e.g., azathiopurine, mycophenolate mofetil), growth factorsignal transduction (e.g., sirolimus, rapamycin), and the T cellinterleukin 2 receptor (e.g., daclizumab, basiliximab). In a particularembodiment, an immunosuppressant agent used in combination withcompositions and methods of the invention includes one or more of thefollowing: adriamycin, azathiopurine, busulfan, cyclophosphamide,cyclosporin A (“CyA”), cytoxin, fludarabine, 5-fluorouracil,methotrexate, mycophenolate mofetil (MOFETIL), nonsteroidalanti-inflammatories (NSAIDs), rapamycin, and tacrolimus (FK506).

The compositions and methods of the present invention can be applied toinflammatory disease for example, inflammation accompanying variousarthritis (for example, rheumatoid arthritis, osteoarthritis),pneumonia, hepatitis (including viral hepatitis), inflammationaccompanying infectious diseases, inflammatory bowel diseases,intestinal enteritis, nephritis (e.g., glomerular nephritis,nephrofibrosis), gastritis, angiitis, pancreatitis, peritonitis,bronchitis, myocarditis, cerebritis, inflammation in postischemicreperfusion injury (myocardial ischemic reperfusion injury),inflammation attributed to immune rejection after transplantation oftissue and organ, burn, various skin inflammation (psoriasis, allergiccontact-type dermatitis, lichen planus), inflammation in multiple organfailure, inflammation after operation of PTCA or PTCR, and inflammationaccompanying arteriosclerosis, and autoimmune thyroiditis.

Compositions of the invention comprising an anti-ICOS antibody withenhanced effector function as an active ingredient may be used toinhibit, treat and/or prevent a variety of diseases, for example, butnot limited to rheumatoid arthritis, multiple sclerosis, autoimmunethyroiditis, allergic contact dermatitis, lichen planus, systemic lupuserythematosus, insulin dependent diabetes mellitus, psoriasis,autoimmune diseases or allergic diseases, delayed allergies mediated bycellular immunity; arthropathies (e.g., rheumatoid arthritis (RA),osteoarthritis (OA)), inflammation (e.g., hepatitis), graft versus hostreaction (GVH reaction), graft versus host disease (GVHD),immunorejection associated with transplantation of tissues (e.g., skin,cornea and bone) or organs (e.g., liver, heart, lung, kidney, pancreas),inflammatory bowel disease, Crohn's disease, ulcerative colitis, andalimentary allergy.

The compositions in accordance with the present invention make itpossible to treat or prevent some inflammations for which varioussteroidal drugs are used as anti-inflammatory drugs, for example,inflammation associated with various arthritides (e.g., rheumatoidarthritis, osteoarthritis), pneumonia, hepatitis (including viralhepatitis), inflammation associated with infectious diseases,inflammatory bowel disease, enteritis, nephritis, glomerular nephritis,inflammation associated with kidney fibrosis, gastritis, vasculitis,pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis,inflammation associated with ischemia-reperfusion injury, myocaridialischemia-reperfusion injury, inflammation associated withimmunorejection after transplantation of tissues or organs, psoriasis,allergic contact dermatitis, lichen planus, inflammation associated withmultiple organ failure, inflammation after operation of PTCA or PTCR,inflammation associated with atherosclerosis, and autoimmunethyroiditis.

5.21. Transplantation

According to certain aspects of the invention, the treatment regimen anddose used with compositions and methods of the invention is chosen basedon a number of factors including, for example, clinical manifestationthat place a patient at risk for developing transplant rejection, orclinical evidence that such a rejection is developing.

The present invention provides compositions, therapeutic formulations,methods and regimens effective to reduce the incidence, severity, orduration of GVHD, a rejection episode, or post-transplantlymphoproliferative disorder. In certain embodiments, compositions andmethods of the invention are effective to attenuate the host response toischemic reperfusion injury of a solid tissue or organ graft. In oneembodiment, compositions and methods of the invention are effective toprolong survival of a graft in a transplant recipient.

The present invention encompasses grafts that are autologous,allogeneic, or xenogeneic to the recipient. The types of graftsencompassed by the invention include tissue and organ grafts, includingbut not limited to, bone marrow grafts, peripheral stem cell grafts,skin grafts, arterial and venous grafts, pancreatic islet cell grafts,and transplants of the kidney, liver, pancreas, thyroid, and heart. Theterms “graft” and “transplant” are used interchangeably herein. In oneembodiment, the autologous graft is a bone marrow graft, an arterialgraft, a venous graft or a skin graft. In one embodiment, the allograftis a bone marrow graft, a corneal graft, a kidney transplant, apancreatic islet cell transplant, or a combined transplant of a kidneyand pancreas. In one embodiment, the graft is a xenograft, wherein thepossible animal donors include, but are not limited to pigs. Thecompositions and methods of the present invention may also be used tosuppress a deleterious immune response to a non-biological graft orimplant, including but not limited to an artificial joint, a stent, or apacemaker device.

Anti-ICOS antibodies, compositions, and methods of the invention may beused to treat or prevent GVHD, rejection, or post-transplantlymphoproliferative disorder without regard to the particularindications initially giving rise to the need for the transplant or theparticular type of tissue transplanted.

Therapeutic formulations and regimens of the present invention aredescribed for treating human subjects diagnosed with autoimmune diseasesor disorders, including but not limited to, rheumatoid arthritis, SLE,ITP, pemphigus-related disorders, diabetes, and scleroderma.

Appropriate treatment regimens can be determined by one of skill in theart for the particular patient or patient population. In particularembodiments, the treatment regimen is a pre-transplant conditioningregimen, a post-transplant maintenance regimen, or post-transplanttreatment regimen for an acute or a chronic rejection. In certainembodiments, the particular regimen is varied for a patient who isassessed as being at a high or intermediate risk of developing arejection response, compared with the regimen for a patient who isassessed as being at a low risk of rejection.

In certain embodiments, the particular regimen is varied according tothe stage of rejection, with more aggressive therapy being indicated forpatients at later stages of rejection. The stages of humoral rejectionmay be classified according to the knowledge and skill in the art. Forexample, the stages of humoral rejection may be classified as one ofstages I to IV according to the following criteria: Stage I LatentResponse, characterized by circulating anti-donor alloantibodies,especially anti-HLA antibodies; Stage II Silent Reaction, characterizedby circulating anti-donor alloantibodies, especially anti-HLAantibodies, and C4d deposition, but without histologic changes or graftdysfunction; Stage III Subclinical Rejection: characterized bycirculating anti-donor alloantibodies, especially anti-HLA antibodies,C4d deposition, and tissue pathology, but without graft dysfunction;Stage 1V Humoral Rejection: characterized by circulating anti-donoralloantibodies, especially anti-HLA antibodies, C4d deposition, tissuepathology, and graft dysfunction.

Anti-ICOS antibodies, compositions and methods of the invention may bepracticed to treat or prevent GVHD, rejection, or post-transplantationlymphoproliferative disorders, either alone or in combination with othertherapeutic agents or treatment regimens. Other therapeutic regimens forthe treatment or prevention of GVHD, rejection, or post-transplantationlymphoproliferative disorders may comprise, for example, one or more ofanti-lymphocyte therapy, steroid therapy, antibody depletion therapy,immunosuppression therapy, and plasmapheresis.

Anti-lymphocyte therapy may comprise the administration to thetransplant recipient of anti-thymocyte globulins, also referred to asthymoglobulin. Anti-lymphocyte therapy may also comprise theadministration of one or more monoclonal antibodies directed against Tcell surface antigens. Examples of such antibodies include, withoutlimitation, OKT3™ (muromonab-CD3), CAMPATH™-1H (alemtuzumab),CAMPATH™-1G, CAMPATH™-1M, SIMULECT™ (basiliximab), and ZENAPAX™(daclizumab). In a specific embodiment, the anti-lymphocyte therapycomprises one or more antibodies directed against B cells, including,without limitation, RITUXAN™ (rituximab).

Steroid therapy may comprise administration to the transplant recipientof one or more steroids selected from the group consisting of cortisol,prednisone, methyl prednisolone, dexamethazone, and indomethacin. One ormore of the steroids may be corticosteroids, including withoutlimitation, cortisol, prednisone, and methylprednisolone.

Antibody depletion therapy may include, for example, administration tothe transplant recipient of intravenous immunoglobulin. Antibodydepletion therapy may also comprise immunoadsorption therapy applied tothe graft ex vivo, prior to transplantation. Immunoadsorption may beaccomplished using any suitable technique, for example, protein Aaffinity, or antibody based affinity techniques using antibodiesdirected against T cell or B cell surface markers such as anti-CD3antibodies, anti-CD19 antibodies, anti-CD₂₀ antibodies, and anti-CD22antibodies.

Immunosuppression therapy may comprise the administration of one or moreimmunosuppressive agents such as inhibitors of cytokine transcription(e.g., cyclosporin A, tacrolimus), nucleotide synthesis (e.g.,azathiopurine, mycophenolate mofetil), growth factor signal transduction(e.g., sirolimus, rapamycin), and the T cell interleukin 2 receptor(e.g., daclizumab, basiliximab). In a particular embodiment, animmunosuppressant agent used in combination with compositions andmethods of the invention includes one or more of the following:adriamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporin A(“CyA”), cytoxin, fludarabine, 5-fluorouracil, methotrexate,mycophenolate mofetil (MOFETIL), nonsteroidal anti-inflammatories(NSAIDs), rapamycin, and tacrolimus (FK506). Immunosuppressive agentsmay also comprise inhibitors of complement, for example, solublecomplement receptor-1, anti-C5 antibody, or a small molecule inhibitorof C1s, for example as described in Buerke et al. (J. Immunol.,167:5375-80 (2001).

In one embodiment, compositions and methods of the invention are used incombination with one or more therapeutic regimens for suppressingrejection, including, without limitation, tacrolimus and mycophenolatemofetil therapy, immunoadsorption, intravenous immunoglobulin therapy,and plasmapheresis.

5.22. Inflammatory Disorder

Anti-ICOS antibodies of the invention may be administered to a subjectin need thereof to prevent, manage, treat or ameliorate an inflammatorydisorder (e.g., asthma) or one or more symptoms thereof. Compositions ofthe invention may also be administered in combination with one or moreother therapies, preferably therapies useful for the prevention,management, treatment or amelioration of an inflammatory disorder(including, but not limited to the prophylactic or therapeutic agentslisted herein) to a subject in need thereof to prevent, manage, treat orameliorate an inflammatory disorder or one or more symptoms thereof. Ina specific embodiment, the invention provides a method of preventing,managing, treating or ameliorating an inflammatory disorder or one ormore symptoms thereof, said method comprising administering to a subjectin need thereof a dose of a prophylactically or therapeuticallyeffective amount of an anti-ICOS antibody of the invention. In anotherembodiment, the invention provides a method of preventing, managing,treating or ameliorating an inflammatory disorder or one or moresymptoms thereof, said method comprising administering to a subject inneed thereof a dose of a prophylactically or therapeutically effectiveamount of an effector function enhanced anti-ICOS antibody of theinvention and a dose of a prophylactically or therapeutically effectiveamount of one or more therapies (e.g., prophylactic or therapeuticagents) other than antibodies (including antibody fragments thereof)that immunospecifically bind to an ICOS polypeptide.

The invention provides methods for managing, treating or amelioratingone or more symptoms of an inflammatory disorder in a subject refractoryto conventional therapies (e.g., methotrexate and a TNF-alpha antagonist(e.g., REMICADE™ or ENBREL™)) for such an inflammatory disorder, saidmethods comprising administering to said subject a dose of aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention. The inventionalso provides methods for managing, treating or ameliorating one or moresymptoms of an inflammatory disorder in a subject refractory to existingsingle agent therapies for such an inflammatory disorder, said methodscomprising administering to said subject a dose of a prophylactically ortherapeutically effective amount of an effector function enhancedanti-ICOS antibody of the invention and a dose of a prophylactically ortherapeutically effective amount of one or more therapies (e.g.,prophylactic or therapeutic agents) other than antibodies (includingantibody fragments thereof) that immunospecifically bind to an ICOSpolypeptide. The invention also provides methods for managing ortreating an inflammatory disorder by administering an effector functionenhanced anti-ICOS antibody of the invention in combination with anyother treatment to patients who have proven refractory to othertreatments but are no longer on these treatments. The invention alsoprovides alternative methods for the treatment of an inflammatorydisorder where another therapy has proven or may prove too toxic, i.e.,results in unacceptable or unbearable side effects, for the subjectbeing treated. For example, a composition of the invention may beadministered to a subject, wherein the subject is refractory to a TNFantagonist or methotrexate. Further, the invention provides methods forpreventing the recurrence of an inflammatory disorder in patients thathave been treated and have no disease activity by administering aneffector function enhanced anti-ICOS antibody of the invention.

Inflammatory disorders that can be treated by the methods encompassed bythe invention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, osteoarthritis, spondyloarthropathies (e.g., psoriaticarthritis, ankylosing spondylitis, Reiter's Syndrome (reactivearthritis), inflammatory osteolysis, Wilson's disease and chronicinflammation resulting from chronic viral or bacteria infections. Asdescribed herein, some autoimmune disorders are associated with aninflammatory condition.

Anti-inflammatory therapies and their dosages, routes of administrationand recommended usage are known in the art and have been described insuch literature as the Physician's Desk Reference (61th ed., 2007).

5.22.1.Anti-Inflammatory Therapies

The present invention provides methods of preventing, managing, treatingor ameliorating an inflammatory disorder or one or more symptomsthereof, said methods comprising administering to a subject in needthereof an effector function enhanced anti-ICOS antibody of theinvention and one or more therapies (e.g., prophylactic or therapeuticagents other than antibodies (including antibody fragments thereof) thatimmunospecifically bind to an ICOS polypeptide. Any agent or therapywhich is known to be useful, or which has been used or is currentlybeing used for the prevention, management, treatment or amelioration ofan inflammatory disorder or one or more symptoms thereof can be used incombination with an effector function enhanced anti-ICOS antibody of theinvention in accordance with the invention described herein.

Any anti-inflammatory agent, including agents useful in therapies forinflammatory disorders, well-known to one of skill in the art can beused in the compositions and methods of the invention. Non-limitingexamples of anti-inflammatory agents include non-steroidalanti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs,anticholinergics (e.g., atropine sulfate, atropine methylnitrate, andipratropium bromide (ATROVENT™)), beta2-agonists (e.g., abuterol(VENTOLIN™ and PROVENTIL™), bitolterol (TORNALATE™), levalbuterol(XOPONEX™), metaproterenol (ALUPENT™), pirbuterol (MAXAIR™), terbutlaine(BRETHAIRE™ and BRETHINE™), albuterol (PROVENTIL™, REPETABS™, andVOLMAX™), formoterol (FORADIL AEROLIZER™), and salmeterol (SEREVEN™ andSEREVEN™ DISKUS™)), and methylxanthines (e.g., theophylline (UNIPHYL™,THEO-DUR™, SLO-BID™, AND TEHO-42™)). Examples of NSAIDs include, but arenot limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac(VOLTAREN™), etodolac (LODINE™), fenoprofen (NALFON™), indomethacin(INDOCIN™), ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone(RELAFEN™), sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib(VIOXX™), naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) andnabumetone (RELAFEN™). Such NSAIDs function by inhibiting acyclooxygenase enzyme (e.g., COX-1 and/or COX-2). Examples of steroidalanti-inflammatory drugs include, but are not limited to,glucocorticoids, dexamethasone (DECADRON™), corticosteroids (e.g.,methylprednisolone (MEDROL™)), cortisone, hydrocortisone, prednisone(PREDNISONE™ and DELTASONE™), prednisolone (PRELONE™ and PEDIAPRED™),triamcinolone, azulfidine, and inhibitors of eicosanoids (e.g.,prostaglandins, thromboxanes, and leukotrienes).

In one embodiment, an effective amount of one or more compositions ofthe invention is administered in combination with a mast cell proteaseinhibitor to a subject at risk of or with an inflammatory disorder. Inanother embodiment, the mast cell protease inhibitor is a tryptasekinase inhibitor, such as, but not limited to GW-45, GW-58, andgenisteine. In a specific embodiment, the mast cell protease inhibitoris phosphatidylinositide-3′ (PI3)-kinase inhibitors, such as, but notlimited to calphostin C. In another embodiment, the mast cell proteaseinhibitor is a protein kinase inhibitor such as, but not limited tostaurosporine. In one embodiment, the mast cell protease inhibitor isadministered locally to the affected area.

Specific examples of immunomodulatory agents which can be administeredin combination with an effector function enhanced anti-ICOS antibody ofthe invention to a subject with an inflammatory disorder include, butare not limited to, methothrexate, leflunomide, cyclophosphamide,cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics(e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide),anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g.,cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthocloneand OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion(Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)),anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate),anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies(e.g., MEDI-507 (MedImmune, Inc., International Publication Nos. WO02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC));anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies,anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)),anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-alpha antibodies,anti-IL-1beta antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies(e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies));CTLA4-immunoglobulin; LFA-3TIP (Biogen, International Publication No. WO93/08656 and U.S. Pat. No. 6,162,432); soluble cytokine receptors (e.g.,the extracellular domain of a TNF-alpha receptor or a fragment thereof,the extracellular domain of an IL-1beta receptor or a fragment thereof,and the extracellular domain of an IL-6 receptor or a fragment thereof);cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-alpha,TNF-beta, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and GM-CSF); andanti-cytokine antibodies (e.g., anti-IL-2 antibodies, anti-IL-4antibodies, anti-IL-6 antibodies, anti-IL-9 antibodies, anti-IL-10antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-IL17antibodies, anti-TNF-alpha antibodies, and anti-IFN-gamma antibodies).

Any TNF-alpha antagonist well-known to one of skill in the art can beused in the compositions and methods of the invention. Non-limitingexamples of TNF-alpha antagonists which can be administered incombination with an effector function enhanced anti-ICOS antibody of theinvention to a subject with an inflammatory disorder include proteins,polypeptides, peptides, fusion proteins, antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments,F(ab)₂ fragments, and antigen-binding fragments thereof) such asantibodies that immunospecifically bind to TNF-alpha, nucleic acidmolecules (e.g., antisense molecules or triple helices), organicmolecules, inorganic molecules, and small molecules that blocks,reduces, inhibits or neutralizes the function, activity and/orexpression of TNF-alpha. In various embodiments, a TNF-alpha antagonistreduces the function, activity and/or expression of TNF-alpha by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99% relative to acontrol such as phosphate buffered saline (PBS). Examples of antibodiesthat immunospecifically bind to TNF-alpha include, but are not limitedto, infliximab (REMICADE™; Centacor), D2E7 (Abbott Laboratories/KnollPharmaceuticals Co., Mt. Olive, N.J.), CDP571 which is also known asHUMICADE™ and CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), andTN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91:2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA89:7375-7379). The present invention also encompasses the use ofantibodies that immunospecifically bind to TNF-alpha disclosed in thefollowing U.S. patents in the compositions and methods of the invention:U.S. Pat. Nos. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380;5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272;5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452;5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and6,171,787; each of which are herein incorporated by reference in theirentirety. Examples of soluble TNF-alpha receptors include, but are notlimited to, sTNF-R1 (Amgen), etanercept (ENBREL™; Immunex) and its rathomolog RENBREL™, soluble inhibitors of TNF-alpha derived from TNFrI,TNFrII (Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87:8331-8335),and TNF-alpha Inh (Seckinger et al, 1990, Proc. Natl. Acad. Sci. USA87:5188-5192).

Other TNF-alpha antagonists encompassed by the invention include, butare not limited to, IL-10, which is known to block TNF-alpha productionvia interferon gamma-activated macrophages (Oswald et al. 1992, Proc.Natl. Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991,Proc. Natl. Acad. Sci. USA 88:10535-10539), the murine product TBP-1(Serono/Yeda), the vaccine CytoTAb (Protherics), antisense molecule104838 (ISIS), the peptide RDP-58 (SangStat), thalidomide (Celgene),CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207(AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469(Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx), Thiazolopyrim(Dynavax), auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals),quinacrine (mepacrine dichlorohydrate), tenidap (Enablex), Melanin(Large Scale Biological), and anti-p38 MAPK agents by Uriach.

Non-limiting examples of anti-inflammatory agents which can beadministered in combination with an effector function enhanced anti-ICOSantibody of the invention to a subject with an inflammatory disorderinclude non-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

In specific embodiments, patients with osteoarthritis are administered aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith other agents or therapies useful for osteoarthritis prevention,treatment, management or amelioration including but not limited to:analgesics (non-limiting examples are acetaminophen, in a dose up to4000 mg/d; phenacetin; and tramadol, in a daily dose in the range of 200to 300 mg); NSAIDs (non-limiting examples include but not limited to,aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate,nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac,and tolmetin. Low dose NSAIDs are preferred, e.g., ibuprofen at 1200mg/d, naproxen at 500 mg/d. A gastroprotective agent, e.g., misoprostol,famotidine or omeprazole, is preferred to use concurrently with aNSAID); nonacetylated salicylates including but not limited tosalsalate; cyclooxygenase (Cox)-2-specific inhibitors (CSIs), includingbut not limited to, celecoxib and rofecoxib; intra- or periarticularinjection of a depot glucocorticoid preparation; intra-articularinjection of hyaluronic acid; capsaicin cream; copious irrigation of theosteroarthritis knee to flush out fibrin, cartilage shards and otherdebris; and joint replacement surgery. Compositions and methods of theinvention can also be used in combination with other nonpharmacologicmeasures in prevention, treatment, management and amelioration ofosteoarthritis including but not limited to: reduction of joint loading(non-limiting examples are correction of poor posture, support forexcessive lumbar lordosis, avoid excessive loading of the involvedjoint, avoid prolonged standing, kneeling and squatting); application ofheat to the affected joint; aerobic exercise and other physicaltherapies.

In specific embodiments, patients with rheumatoid arthritis areadministered a prophylactically or therapeutically effective amount ofan effector function enhanced anti-ICOS antibody of the invention incombination with other agents or therapies useful in prevention,treatment, management and amelioration of rheumatoid arthritis includingbut not limited to: NSAIDs (non-limiting examples include but notlimited to, aspirin, diflunisal, diclofenac, etodolac, fenamates,fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen,methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone,piroxicam, sulindac, and tolmetin.); analgesics (non-limiting examplesare acetaminophen, phenacetin and tramadol); CSIs including but notlimited to, celecoxib and rofecoxib; glucocorticoids (preferablylow-dose oral glucocorticoids, e.g., <7.5 mg/d prednisone, or monthlypulses with high-dose glucocorticoids, or intraarticularglucocorticoids); disease-modifying antirheumatic drugs (DMARDs)including but not limited to, methotrexate (preferably givenintermittent low dose, e.g., 7.5-30 mg once weekly), gold compounds(e.g., gold salts), D-penicillamine, the antimalarials (e.g.,chloroquine), and sulfasalazine; TNF-alpha neutralizing agents includingbut not limited to, etanercept and infliximab; immunosuppressive andcytotoxic agents (examples include but not limited to, azathioprine,leflunomide, cyclosporine, and cyclophosphamide), and surgery (examplesinclude but not limited to, arthroplasties, total joint replacement,reconstructive hand surgery, open or arthroscopic synovectomy, and earlytenosynovectomy of the wrist). The compositions and methods of theinvention may also be used in combination with other measures inprevention, treatment, management and amelioration of the rheumatoidarthritis including but not limited to: rest, splinting to reduceunwanted motion of inflamed joint, exercise, used of a variety oforthotic and assistive devices, and other physical therapies. Thecompositions and methods of the invention may also be used incombination with some nontraditional approaches in prevention,treatment, management and amelioration of rheumatoid arthritis includingbut not limited to, diets (e.g., substituting omega-3 fatty acids suchas eicosapentaenoic acid found in certain fish oils for dietary omega-6essential fatty acids found in meat), vaccines, hormones and topicalpreparations.

In specific embodiments, patients with chronic obstructive pulmonarydisease (COPD) are administered a prophylactically or therapeuticallyeffective amount of an effector function enhanced anti-ICOS antibody ofthe invention in combination with other agents or therapies useful inprevention, treatment, management and amelioration of COPD including butnot limited to: bronchodilators including but not limited to, short- andlong-acting beta2-adrenergic agonists (examples of short-acting beta2agonist include but not limited to, albuterol, pirbuterol, terbutaline,and metaproterenol; examples of long-acting beta2 agonist include butnot limited to, oral sustained-release albuterol and inhaledsalmeterol), anticholinergics (examples include but not limited toipratropium bromide), and theophylline and its derivatives (therapeuticrange for theophylline is preferably 10-20 .mu.g/mL); glucocorticoids;exogenous alpha1AT (e.g., alpha1AT derived from pooled human plasmaadministered intravenously in a weekly dose of 60 mg/kg); oxygen; lungtransplantation; lung volume reduction surgery; endotracheal intubation,ventilation support; yearly influenza vaccine and pneumococcalvaccination with 23-valent polysaccharide; exercise; and smokingcessation.

In specific embodiments, patients with asthma are administered aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith an effective amount of one or more other agents useful for asthmatherapy. Non-limiting examples of such agents include adrenergicstimulants (e.g., catecholamines (e.g., epinephrine, isoproterenol, andisoetharine), resorcinols (e.g., metaproterenol, terbutaline, andfenoterol), and saligenins (e.g., salbutamol)), adrenocorticoids,blucocorticoids, corticosteroids (e.g., beclomethadonse, budesonide,flunisolide, fluticasone, triamcinolone, methylprednisolone,prednisolone, and prednisone), other steroids, beta2-agonists (e.g.,albtuerol, bitolterol, fenoterol, isoetharine, metaproterenol,pirbuterol, salbutamol, terbutaline, formoterol, salmeterol, andalbutamol terbutaline), anti-cholinergics (e.g., ipratropium bromide andoxitropium bromide), IL-4 antagonists (including antibodies), IL-5antagonists (including antibodies), IL-9 antagonists (includingantibodies), IL-13 antagonists (including antibodies), IL_(—)17antagonists (including antibodies), PDE4-inhibitor, NF-Kappa-betainhibitor, VLA-4 inhibitor, CpG, anti-CD23, selectin antagonists (TBC1269), mast cell protease inhibitors (e.g., tryptase kinase inhibitors(e.g., GW-45, GW-58, and genisteine), phosphatidylinositide-3′(PI3)-kinase inhibitors (e.g., calphostin C), and other kinaseinhibitors (e.g., staurosporine) (see Temkin et al., 2002 J Immunol169(5):2662-2669; Vosseller et al., 1997 Mol. Biol. Cell 8(5):909-922;and Nagai et al., 1995 Biochem Biophys Res Commun 208(2):576-581)), a C3receptor antagonists (including antibodies), immunosuppressant agents(e.g., methotrexate and gold salts), mast cell modulators (e.g.,cromolyn sodium (INTAL™) and nedocromil sodium (TILADE™)), and mucolyticagents (e.g., acetylcysteine)). In a specific embodiment, theanti-inflammatory agent is a leukotriene inhibitor (e.g., montelukast(SINGULAIR™), zafirlukast (ACCOLATE™), pranlukast (ONON™), or zileuton(ZYFLO™)).

In specific embodiments, patients with allergy are administered aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith an effective amount of one or more other agents useful for allergytherapy. Non-limiting examples of such agents include antimediator drugs(e.g., antihistamine), corticosteroids, decongestants, sympathomimeticdrugs (e.g., alpha-adrenergic and .beta-adrenergic drugs), TNX901 (Leunget al., N Engl J Med 348(11):986-993 (2003)), IgE antagonists (e.g.,antibodies rhuMAb-E25 omalizumab (see Finn et al., 2003 J Allergy ClinImmuno 111(2):278-284; Corren et al., 2003 J Allergy Clin Immuno111(1):87-90; Busse and Neaville, 2001 Curr Opin Allergy Clin Immuno1(1):105-108; and Tang and Powell, 2001, Eur J Pediatr 160(12):696-704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int Arch AllergyImmuno 128(1):24-32), and mAB Hu-901 (see van Neerven et al., 2001 IntArch Allergy Immuno 124(1-3):400), theophylline and its derivatives,glucocorticoids, and immunotherapies (e.g., repeated long-term injectionof allergen, short course desensitization, and venom immunotherapy).

5.23. Autoimmune Disease

According to certain aspects of the invention, the treatment regimen anddose used with compositions and methods of the invention is chosen basedon a number of factors including, but not limited to, the stage of theautoimmune disease or disorder being treated. Appropriate treatmentregimens can be determined by one of skill in the art for particularstages of an autoimmune disease or disorder in a patient or patientpopulation. Dose response curves can be generated using standardprotocols in the art in order to determine the effective amount ofcompositions of the invention for treating patients having differentstages of a autoimmune disease or disorder. In general, patients havingmore activity of a autoimmune disease or disorder will require higherdoses and/or more frequent doses which may be administered over longerperiods of time in comparison to patients having less activity of anautoimmune disease or disorder.

Anti-ICOS antibodies, compositions and methods may be practiced to treatan autoimmune disease or disorder. The term “autoimmune disease ordisorder” refers to a condition in a subject characterized by cellular,tissue and/or organ injury caused by an immunologic reaction of thesubject to its own cells, tissues and/or organs. The term “inflammatorydisease” is used interchangeably with the term “inflammatory disorder”to refer to a condition in a subject characterized by inflammation,including, but not limited to chronic inflammation. Autoimmune disordersmay or may not be associated with inflammation. Moreover, inflammationmay or may not be caused by an autoimmune disorder. Thus, certaindisorders may be characterized as both autoimmune and inflammatorydisorders. Exemplary autoimmune diseases or disorders include, but arenot limited to: alopecia greata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunediseases of the adrenal gland, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, diabetes, eosinophilic fascites,fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonlein purpura,idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Meniere's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus-related disorders (e.g., pemphigusvulgaris), pernicious anemia, polyarteritis nodosa, polychrondritis,polyglandular syndromes, polymyalgia rheumatica, polymyositis anddermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis,psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter'ssyndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren'ssyndrome, stiff-man syndrome, systemic lupus erythematosis (SLE),Sweet's syndrome, Still's disease, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, graft versus host disease,urticaria, Vogt-Koyanagi-Hareda syndrome and chronic inflammationresulting from chronic viral or bacteria infections.

5.23.1.Autoimmune Disorder Treatment

An effector function enhanced anti-ICOS antibody of the invention may beadministered to a subject in need thereof to prevent, manage, treat orameliorate an autoimmune disorder or one or more symptoms thereof.Compositions of the invention may also be administered in combinationwith one or more other therapies, preferably therapies useful for theprevention, management or treatment of an autoimmune disorder(including, but not limited to the prophylactic or therapeutic agents)to a subject in need thereof to prevent, manage, treat or ameliorate anautoimmune disorder or one or more symptoms thereof. In a specificembodiment, the invention provides a method of preventing, managing,treating or ameliorating an autoimmune disorder or one or more symptomsthereof, said method comprising administering to a subject in needthereof a dose of a prophylactically or therapeutically effective amountof an effector function enhanced anti-ICOS antibody of the invention. Inanother embodiment, the invention provides a method of preventing,managing, treating or ameliorating an autoimmune disorder or one or moresymptoms thereof, said method comprising administering to a subject inneed thereof a dose of a prophylactically or therapeutically effectiveamount of an effector function enhanced anti-ICOS antibody of theinvention and a dose of a prophylactically or therapeutically effectiveamount of one or more therapies (e.g., prophylactic or therapeuticagents) other than antibodies (including antibody fragments thereof)that immunospecifically bind to an ICOS polypeptide.

The invention provides methods for managing, treating or ameliorating anautoimmune disorder or one or more symptoms thereof in a subjectrefractory to conventional therapies for such an autoimmune disorder,said methods comprising administering to said subject a dose of aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention. The inventionalso provides methods for managing, treating or ameliorating anautoimmune disorder or one or more symptoms thereof in a subjectrefractory to existing single agent therapies for such an autoimmunedisorder, said methods comprising administering to said subject a doseof a prophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention and a dose of aprophylactically or therapeutically effective amount of one or moretherapies (e.g., prophylactic or therapeutic agents) other thanantibodies (including antibody fragments thereof) thatimmunospecifically bind to an ICOS polypeptide. The invention alsoprovides methods for managing, treating or ameliorating an autoimmunedisorder or one or more symptoms thereof by administering an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith any other treatment to patients who have proven refractory to othertreatments but are no longer on these treatments. The invention alsoprovides alternative methods for the management or treatment of anautoimmune disorder where another therapy has proven or may prove tootoxic, i.e., results in unacceptable or unbearable side effects, for thesubject being treated. Particularly, the invention provides alternativemethods for the management or treatment of an autoimmune disorder wherethe patient is refractory to other therapies. Further, the inventionprovides methods for preventing the recurrence of an autoimmune disorderin patients that have been treated and have no disease activity byadministering an effector function enhanced anti-ICOS antibody of theinvention.

Examples of autoimmune disorders that can be treated by the methods ofthe invention include, but are not limited to, alopecia greata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune oophoritis and orchitis,autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Mnire's disease, mixed connective tissue disease, multiplesclerosis, type 1 or immune-mediated diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld'sphenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupuserythematosus, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis.

Autoimmune therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (61th ed., 2007).

5.23.2.Autoimmune Disorder Therapies

The present invention provides methods of preventing, managing, treatingor ameliorating an autoimmune disorder or one or more symptoms thereof,said methods comprising administering to a subject in need thereof aneffector function enhanced anti-ICOS antibody of the invention and oneor more therapies (e.g., prophylactic or therapeutic agents) other thanantibodies (including antibody fragments thereof) thatimmunospecifically bind to an ICOS polypeptide. Any agent or therapywhich is known to be useful, or which has been used or is currentlybeing used for the prevention, management, treatment or amelioration ofan autoimmune disorder or one or more symptoms thereof can be used incombination with an effector function enhanced anti-ICOS antibody of theinvention in accordance with the invention described herein. Examples ofsuch agents include, but are not limited to, immunomodulatory agents,anti-inflammatory agents and TNF-alpha antagonists. Specific examples ofimmunomodulatory agents, anti-inflammatory agents and TNF-alphaantagonists which can be used in combination with an effector functionenhanced anti-ICOS antibody of the invention for the prevention,management, treatment or amelioration of an autoimmune disorder aredisclosed herein.

In specific embodiments, patients with multiple sclerosis (MS) areadministered a prophylactically or therapeutically effective amount ofan effector function enhanced anti-ICOS antibody of the invention incombination with other agents or therapies useful in prevention,treatment, management and amelioration of MS including but not limitedto: IFN-beta1b (Betaseron) (e.g., 8.0 million international unites (MIU)is administered by subcutaneous injection every other day); IFN-beta1a(Avonex) (e.g., 6.0 MIU is administered by intramuscular injection onceevery week); glatiramer acetate (Copaxone) (e.g., 20 mg is administeredby subcutaneous injection every day); mitoxantrone (e.g., 12 mg/m² isadministered by intravenous infusion every third month); azathioprine(e.g., 2-3 mg/kg body weight is administered orally each day);methotrexate (e.g., 7.5 mg is administered orally once each week);cyclophosphamide; intravenous immunoglobulin (e.g., 0.15-0.2 g/kg bodyweight administered monthly for up to 2 years); glucocorticoids;methylprednisolone (e.g., administered in bimonthly cycles at highdoses); 2-chlorodeoxyadenosine (cladribine); baclofen (e.g., 15 to 80mg/d in divided doses, or orally in higher doses up to 240 mg/d, orintrathecally via an indwelling catheter); cycloenzaprine hydrochloride(e.g., 5-10 mg bid or tid); clonazepam (e.g., 0.5 to 1.0 mg tid,including bedtime dose); clonidine hydrochloride (e.g., 0.1 to 0.2 mgtid, including a bedtime dose); carbamazepine (e.g., 100-1200 mg/d individed, escalating doses); gabapentin (e.g., 300-3600 mg/d); dilantin(e.g., 300-400 mg/d); amitriptyline (e.g., 25-150 mg/d); baclofen (e.g.,10-80 mg/d); primidone (e.g., 125-250 mg bid or tid); ondansetron (e.g.,4 to 8 mg bid or tid); isoniazid (e.g., up to 1200 mg in divided doses);oxybutynin (e.g., 5 mg bid or tid); tolterodine (e.g., 1-2 mg bid);propantheline (e.g., 7.5 to 15 mg qid); bethanecol (e.g., 10-50 mg tidor qid); terazosin hydrochloride (e.g., 1-5 mg at bedtime); sildenafilcitrate (e.g., 50-100 mg po prn); amantading (e.g., 100 mg bid);pemoline (e.g., 37.5 mg bid); high dose vitamins; calcium orotate;gancyclovir; antibiotic; and plasma exchange.

In specific embodiments, patients with psoriasis are administered aprophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith other agents or therapies useful in prevention, treatment,management and amelioration of psoriasis including but not limited to:topical steroid cream or ointment; tar (examples including but notlimited to, Estar, Psorigel, Fototar cream, and LCD 10% in Nutradermlotion or mixed directly with triamcinolone 0.1% cream); occlusion;topical vitamin D analogue (a non-limiting example is calcipotrieneointment); ultraviolet light; PUVA (psoralen plus ultraviolet A);methotrexate (e.g., up to 25 mg once weekly or in divided doses every 12hours for three doses once a week); synthetic retinoid (a non-limitingexamples is etretinate, e.g., in dosage of 0.5-1 mg/kg/d);immunomodulatory therapy (a non-limiting example is cyclosporine);sulfasalazine (e.g., in dosages of 1 g three times daily).

In specific embodiments, patients with Crohn's disease are administereda prophylactically or therapeutically effective amount of an effectorfunction enhanced anti-ICOS antibody of the invention in combinationwith other agents or therapies useful in prevention, treatment,management and amelioration of Crohn's disease including but not limitedto: antidiarrheals (e.g., loperamide 2-4 mg up to 4 times a day,diphenoxylate with atropine 1 tablet up to 4 times a day, tincture ofopium 8-15 drops up to 4 times a day, cholestyramine 2-4 g or colestipol5 g once or twice daily), antispasmodics (e.g., propantheline 15 mg,dicyclomine 10-20 mg, or hyoscyamine 0.125 mg given before meals),5-aminosalicylic acid agents (e.g., sulfasalazine 1.5-2 g twice daily,mesalamine (ASACOL™) and its slow release form (PENTASA™), especially athigh dosages, e.g., PENTASA™ 1 g four times daily and ASACOL™ 0.8-1.2 gfour times daily), corticosteroids, immunomodulatory drugs (e.g.,azathioprine (1-2 mg/kg), mercaptopurine (50-100 mg), cyclosporine, andmethotrexate), antibiotics, TNF inhibitors (e.g., inflixmab(REMICADE™)), immunosuppressive agents (e.g., tacrolimus, mycophenolatemofetil, and thalidomide), anti-inflammatory cytokines (e.g., IL-10 andIL-11), nutritional therapies, enteral therapy with elemental diets(e.g., Vivonex for 4 weeks), and total parenteral nutrition.

In specific embodiments, patients with lupus erythematosus areadministered a prophylactically or therapeutically effective amount ofan effector function enhanced anti-ICOS antibody of the invention incombination with other agents or therapies useful in prevention,treatment, management and amelioration of lupus erythematosus includingbut not limited to: antimalarials (including but not limited to,hydroxychloroquine); glucocorticoids (e.g., low dose, high dose, orhigh-dose intravenous pulse therapy can be used); immunosuppressiveagents (including but not limited to, cyclophosphamide, chlorambucil,and azanthioprine); cytotoxic agents (including but not limited tomethotrexate and mycophenolate mofetil); androgenic steroids (includingbut not limited to danazol); and anticoagulants (including but notlimited to warfarin).

The antibody formulations of the invention or combination therapies ofthe invention may be used as the first, second, third, fourth, or fifththerapy to prevent, manage, treat, and/or ameliorate an autoimmunedisorder or one or more symptom thereof. The invention also includesmethods of preventing, treating, managing, and/or ameliorating anautoimmune disorder or one or more symptoms thereof in a patientundergoing therapies for other disease or disorders. The inventionencompasses methods of preventing, managing, treating, and/orameliorating an autoimmune disorder or one or more symptoms thereof in apatient before any adverse effects or intolerance to therapies otherthan antibodies of the invention develops. The invention alsoencompasses methods of preventing, treating, managing, and/orameliorating an autoimmune disorder or a symptom thereof in refractorypatients. The invention encompasses methods for preventing, treating,managing, and/or ameliorating a proliferative disorder or a symptomthereof in a patient who has proven refractory to therapies other thanantibodies, compositions, or combination therapies of the invention. Thedetermination of whether a patient is refractory can be made either invivo or in vitro by any method known in the art for assaying theeffectiveness of a treatment of autoimmune disorders, using art-acceptedmeanings of “refractory” such a context. In certain embodiments, apatent with an autoimmune disorder is refractory to a therapy when oneor more symptoms of an autoimmune disorder is not prevented, managed,and/or alleviated. The invention also encompasses methods of preventing,managing, treating, and/or ameliorating an autoimmune disorder or asymptom thereof in patients who are susceptible to adverse reactions toconventional therapies.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating an autoimmune disorder or one or moresymptoms thereof as an alternative to other conventional therapies. Inspecific embodiments, the patient being managed or treated in accordancewith the methods of the invention is refractory to other therapies or issusceptible to adverse reactions from such therapies. The patient may bea person with a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease,patients with broncho-pulmonary dysplasia, patients with congenitalheart disease, patients with cystic fibrosis, patients with acquired orcongenital heart disease, and patients suffering from an infection), aperson with impaired renal or liver function, the elderly, children,infants, infants born prematurely, persons with neuropsychiatricdisorders or those who take psychotropic drugs, persons with historiesof seizures, or persons on medication that would negatively interactwith conventional agents used to prevent, manage, treat, or amelioratean autoimmune disease or disorder.

Autoimmune therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in suchliterature as the Physician's Desk Reference (61th ed., 2007).

5.23.3. Diagnosis of Autoimmune Diseases or Disorders

The diagnosis of an autoimmune disease or disorder is complicated inthat each type of autoimmune disease or disorder manifests differentlyamong patients. This heterogeneity of symptoms means that multiplefactors are typically used to arrive at a clinical diagnosis. Generally,clinicians use factors, such as, but not limited to, the presence ofautoantibodies, elevated cytokine levels, specific organ dysfunction,skin rashes, joint swelling, pain, bone remodeling, and/or loss ofmovement as primarily indicators of an autoimmune disease or disorder.For certain autoimmune diseases or disorders, such as RA and SLE,standards for diagnosis are known in the art. For certain autoimmunediseases or disorders, stages of disease have been characterized and arewell known in the art. These art recognized methods for diagnosingautoimmune diseases and disorders as well as stages of disease andscales of activity and/or severity of disease that are well known in theart can be used to identify patients and patient populations in need oftreatment for an autoimmune disease or disorder using compositions andmethods of the invention.

5.23.4. Clinical Criteria for Diagnosing Autoimmune Diseases orDisorders

Diagnostic criteria for different autoimmune diseases or disorders areknown in the art. Historically, diagnosis is typically based on acombination of physical symptoms. More recently, molecular techniquessuch as gene-expression profiling have been applied to develop moleculardefinitions of autoimmune diseases or disorders. Exemplary methods forclinical diagnosis of particular autoimmune diseases or disorders areprovided below. Other suitable methods will be apparent to those skilledin the art.

In certain embodiments, patients with low levels of autoimmune diseaseactivity or patients with an early stage of an autoimmune disease (fordiseases where stages are recognized) can be identified for treatmentusing anti-ICOS antibody compositions and methods. The early diagnosisof autoimmune disease is difficult due to the general symptoms andoverlap of symptoms among diseases. In such embodiments, a patienttreated at an early stage or with low levels of an autoimmune diseaseactivity has symptoms comprising at least one symptom of an autoimmunedisease or disorder. In related embodiments, a patient treated at anearly stage or with low levels of an autoimmune disease has symptomscomprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15symptoms of an autoimmune disease or disorder. The symptoms may be ofany autoimmune diseases and disorders or a combination thereof. Examplesof autoimmune disease and disorder symptoms are described below.

5.24. Immunotherapeutic Protocols

Anti-ICOS antibody compositions used in the therapeuticregimen/protocols, referred to herein as “anti-ICOS immunotherapy” canbe naked antibodies, immunoconjugates and/or fusion proteins.Compositions of the invention can be used as a single agent therapy orin combination with other therapeutic agents or regimens. Anti-ICOSantibodies or immunoconjugates can be administered prior to,concurrently with, or following the administration of one or moretherapeutic agents. Therapeutic agents that can be used in combinationtherapeutic regimens with compositions of the invention include anysubstance that inhibits or prevents the function of cells and/or causesdestruction of cells. Examples include, but are not limited to,radioactive isotopes, chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

The therapeutic regimens described herein, or any desired treatmentregimen can be tested for efficacy using a transgenic animal model whichexpresses human ICOS antigen in place of native ICOS antigen. Thus, ananti-ICOS antibody treatment regimen can be tested in an animal model todetermine efficacy before administration to a human.

5.25. Anti-ICOS Immunotherapy

In accordance with the present invention “anti-ICOS immunotherapy”encompasses the administration of any of the anti-ICOS antibodies of theinvention in accordance with any therapeutic regimen described herein.Anti-ICOS antibodies can be administered as naked antibodies, orimmunoconjugates or fusion proteins. In one embodiment, a human subjecthaving a T cell-mediated disease or disorder can be treated byadministering an anti-ICOS antibody capable to mediate human ADCC.

Antibodies of IgG1 or IgG3 human isotypes are in some cases preferredfor therapy. However, the IgG2 or IgG4 human isotypes can be used aswell, provided they have the relevant effector function, for examplehuman ADCC. Such effector function can be assessed by measuring theability of the antibody in question to mediate target cell lysis byeffector cells in vitro or in vivo.

In one embodiment, the dose of antibody used should be sufficient todeplete circulating ICOS expressing T cells. Progress of the therapy canbe monitored in the patient by analyzing blood samples. Other signs ofclinical improvement can be used to monitor therapy.

Methods for measuring depletion of ICOS expressing T cells that can beused in connection with compositions and methods of the invention arewell known in the art and include, but are not limited to the followingembodiments. In one embodiment, circulating ICOS expressing T cellsdepletion can be measured with flow cytometry using a reagent other thanan anti-ICOS antibody that binds to ICOS expressing T cells to definethe amount of ICOS expressing T cells. In another embodiment, ICOSexpressing T cell depletion can be measured by immunochemical stainingto identify ICOS expressing T cells. In such embodiments, ICOSexpressing T cells or tissues or serum comprising ICOS expressing Tcells extracted from a patient can be placed on microscope slides,labeled and examined for presence or absence. In related embodiments, acomparison is made between ICOS expressing T cells extracted prior totherapy and after therapy to determine differences in the presence ofICOS expressing T cells.

In embodiments of the invention where an anti-ICOS antibody isadministered as a single agent therapy, the invention contemplates useof different treatment regimens.

According to certain aspects of the invention, an anti-ICOS antibodyused in compositions and methods of the invention, is a naked antibody.In related embodiments, the dose of naked anti-ICOS antibody used is atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5 mg/kg of body weight of a patient. In certainembodiments, the dose of naked anti-ICOS antibody used is at least about1 to 10, 5 to 15, 10 to 20, or 15 to 25 mg/kg of body weight of apatient. In certain embodiments, the dose of naked anti-ICOS antibodyused is at least about 1 to 20, 3 to 15, or 5 to 10 mg/kg of body weightof a patient. In other embodiments, the dose of naked anti-ICOS antibodyused is at least about 5, 6, 7, 8, 9, or 10 mg/kg of body weight of apatient.

In certain embodiments, the dose comprises about 375 mg/m² of anti-ICOSantibody administered weekly for about 1, 2, 3, 4, 5, 6, 7 or 8consecutive weeks. In certain embodiments, the dose is at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/kg of body weightof the patient administered weekly for about 1, 2, 3, 4, 5, 6, 7 or 8consecutive weeks.

The exemplary doses of anti-ICOS antibody described above can beadministered as described herein. In one embodiment, the above doses aresingle dose injections. In other embodiments, the doses are administeredover a period of time. In other embodiments, the doses are administeredmultiple times over a period of time. The period of time may be measuredin days, weeks, or months. Multiple doses of an anti-ICOS antibody canbe administered at intervals suitable to achieve a therapeutic benefitwhile balancing toxic side effects. For example, where multiple dosesare used, it may be preferred to time the intervals to allow forrecovery of the patient's monocyte count prior to the repeat treatmentwith antibody. This dosing regimen will optimize the efficiency oftreatment, since the monocyte population reflects ADCC function in thepatient.

In certain embodiments, compositions of the invention are administeredto a human patient as long as the patient is responsive to therapy. Inother embodiments, compositions of the invention are administered to ahuman patient as long as the patient's disease does not progress. Inrelated embodiments, compositions of the invention are administered to ahuman patient until a patient's disease does not progress or has notprogressed for a period of time, then the patient is not administeredcompositions of the invention unless the disease reoccurs or begins toprogress again. If disease progression stops or reverses, then hepatient will not be administered compositions of the invention untilthat patient relapses, i.e., the disease being treated reoccurs orprogresses. Upon this reoccurrence or progression, the patient can betreated again with the same dosing regimen initially used or using otherdoses described above.

In certain embodiments, compositions of the invention can beadministered as a loading dose followed by multiple lower doses(maintenance doses) over a period of time. In such embodiments, thedoses may be timed and the amount adjusted to maintain effective ICOSexpressing T cell depletion. In certain embodiments, the loading dose isabout 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg/kg of patient body weightand the maintenance dose is at least about 5 to 10 mg/kg of patient bodyweight. In other embodiments, the maintenance dose is administered atintervals of every 7, 10, 14 or 21 days.

5.26. Combination with Chemotherapeutic Agents

Anti-ICOS immunotherapy (using naked antibody, immunoconjugates, orfusion proteins) can be used in conjunction with other therapiesincluding but not limited to, chemotherapy, radioimmunotherapy (RIT),chemotherapy and external beam radiation (combined modality therapy,CMT), or combined modality radioimmunotherapy (CMRIT) alone or incombination, etc. In certain embodiments, an anti-ICOS antibody therapyof the present invention can be administered in conjunction with CHOP(Cyclophosphamide-Hydroxydoxorubicin-Oncovin (vincristine)-Prednisolone)As used herein, the term “administered in conjunction with” means thatan anti-ICOS immunotherapy can be administered before, during, orsubsequent to the other therapy employed.

In certain embodiments, an anti-ICOS immunotherapy is in conjunctionwith a cytotoxic radionuclide or radiotherapeutic isotope. For example,an alpha-emitting isotope such as ²²⁵Ac, ²²⁴Ac, ²¹¹At, ²¹²Bi, ²¹³Bi,²¹²Pb, ²²⁴Ra, or ²²³Ra. The cytotoxic radionuclide may also be abeta-emitting isotope such as ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu,¹⁵³Sm, ¹⁶⁶Ho, or ⁶⁴Cu. Further, the cytotoxic radionuclide may emitAuger and low energy electrons and include the isotopes ¹²⁵I, ¹²³I or⁷⁷Br. In other embodiments the isotope may be ¹⁹⁸Au, ³²P, and the like.In certain embodiments, the amount of the radionuclide administered tothe subject is between about 0.001 mCi/kg and about 10 mCi/kg.

In some embodiments, the amount of the radionuclide administered to thesubject is between about 0.1 mCi/kg and about 1.0 mCi/kg. In otherembodiments, the amount of the radionuclide administered to the subjectis between about 0.005 mCi/kg and 0.1 mCi/kg.

In certain embodiments, an anti-ICOS immunotherapy is in conjunctionwith a chemical toxin or chemotherapeutic agent. The chemical toxin orchemotherapeutic agent may be selected from the group consisting of anenediyne such as calicheamicin and esperamicin; duocarmycin,methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine,mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.

Suitable chemical toxins or chemotherapeutic agents that can be used incombination therapies with an anti-ICOS immunotherapy include members ofthe enediyne family of molecules, such as calicheamicin and esperamicin.Chemical toxins can also be taken from the group consisting ofduocarmycin (see, e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No.4,923,990), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and5-fluorouracil. Examples of chemotherapeutic agents also includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

In other embodiments, for example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) can be used incombination therapies of the invention. CVB is a regimen used to treatnon-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51:18 (1993).Other suitable combination chemotherapeutic regimens are well-known tothose of skill in the art. See, for example, Freedman et al.,“Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rd Edition,Holland et al (eds.), pp. 2028-2068 (Lea & Febiger 1993). As anillustration, first generation chemotherapeutic regimens for treatmentof intermediate-grade non-Hodgkin's lymphoma include C-MOPP(cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone). A usefulsecond generation chemotherapeutic regimen is m-BACOD (methotrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate and brostatin-1. In a multimodal therapy, both chemotherapeuticdrugs and cytokines are co-administered with an antibody,immunoconjugate or fusion protein according to the present invention.The cytokines, chemotherapeutic drugs and antibody, immunoconjugate orfusion protein can be administered in any order, or together.

Other toxins that may be used in compositions and methods of theinvention include poisonous lectins, plant toxins such as ricin, abrin,modeccin, botulina and diphtheria toxins. Of course, combinations of thevarious toxins could also be coupled to one antibody molecule therebyaccommodating variable cytotoxicity. Illustrative of toxins which aresuitably employed in combination therapies of the invention are ricin,abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg et al., Cancer Journal for Clinicians 44:43(1994). Enzymatically active toxins and fragments thereof which can beused include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

Suitable toxins and chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985). Other suitable toxins and/orchemotherapeutic agents are known to those of skill in the art.

An anti-ICOS immunotherapy of the present invention may also be inconjunction with a prodrug-activating enzyme which converts a prodrug(e.g., a peptidyl chemotherapeutic agent, see, WO81/01145) to an activeanti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278. The enzyme component of such combinations includes any enzymecapable of acting on a prodrug in such a way so as to covert it into itsmore active, cytotoxic form. The term “prodrug” as used in thisapplication refers to a precursor or derivative form of apharmaceutically active substance that is less cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into the more active parent form. See, e.g.,Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, HumanaPress (1985). Prodrugs that can be used in combination with anti-ICOSantibodies include, but are not limited to, phosphate-containingprodrugs, thiophosphate-containing prodrugs, sulfate-containingprodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, α-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

In certain embodiments, administration of compositions and methods ofthe invention may enable the postponement of toxic therapy and may helpavoid unnecessary side effects and the risks of complications associatedwith chemotherapy and delay development of resistance to chemotherapy.In certain embodiments, toxic therapies and/or resistance to toxictherapies is delayed in patients administered compositions and methodsof the invention delay for up to about 6 months, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 years.

5.27. Combination with Therapeutic Antibodies

An anti-ICOS immunotherapy described herein may be administered incombination with other antibodies, including, but not limited to,anti-CD19 mAb, anti-CD52 mAb, anti-CD22 antibody, and anti-CD20antibodies, such as RITUXAN™ (C2B8; RITUXIMAB™; IDEC Pharmaceuticals).Other examples of therapeutic antibodies that can be used in combinationwith antibodies of the invention or used in compositions of theinvention include, but are not limited to, HERCEPTIN™ (Trastuzumab;Genentech), MYLOTARG™ (Gemtuzumab ozogamicin; Wyeth Pharmaceuticals),CAMPATH™ (Alemtuzumab; Berlex), ZEVALIN™ (Ipritumomab tiuxetan; BiogenIdec), BEXXAR™ (Tositumomab; GlaxoSmithKline Corixa), ERBITUX™(Cetuximab; Imclone), and AVASTIN™ (Bevacizumab; Genentech).

5.28. Combination Compounds that Enhance Monocyte or Macrophage Function

In certain embodiments of methods of the invention, a compound thatenhances monocyte or macrophage function (e.g., at least about 25%, 50%,75%, 85%, 90%, 95% or more) can be used in conjunction with an anti-ICOSimmunotherapy. Such compounds are known in the art and include, withoutlimitation, cytokines such as interleukins (e.g., IL-12), andinterferons (e.g., alpha or gamma interferon).

The compound that enhances monocyte or macrophage function orenhancement can be formulated in the same pharmaceutical composition asthe antibody, immunoconjugate or antigen-binding fragment. Whenadministered separately, the antibody/fragment and the compound can beadministered concurrently (within a period of hours of each other), canbe administered during the same course of therapy, or can beadministered sequentially (i.e., the patient first receives a course ofthe antibody/fragment treatment and then a course of the compound thatenhances macrophage/monocyte function or vice versa). In suchembodiments, the compound that enhances monocyte or macrophage functionis administered to the human subject prior to, concurrently with, orfollowing treatment with other therapeutic regimens and/or compositionsof the invention. In one embodiment, the human subject has a bloodleukocyte, monocyte, neutrophil, lymphocyte, and/or basophil count thatis within the normal range for humans. Normal ranges for human bloodleukocytes (total) is about 3.5-about 10.5 (10⁹/L). Normal ranges forhuman blood neutrophils is about 1.7-about 7.0 (10⁹/L), monocytes isabout 0.3-about 0.9 (10⁹/L), lymphocytes is about 0.9-about 2.9 (10⁹/L),basophils is about 0-about 0.3 (10⁹/L), and eosinophils is about0.05-about 0.5 (10⁹/L). In other embodiments, the human subject has ablood leukocyte count that is less than the normal range for humans, forexample at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or0.8 (10⁹/L) leukocytes.

5.29. Combination with Immunoregulatory Agents

The anti-ICOS immunotherapy of the present invention may also be inconjunction with an immunoregulatory agent. The term “immunoregulatoryagent” as used herein for combination therapy refers to substances thatact to suppress, mask, or enhance the immune system of the host.

Examples of immunomodulatory agents include, but are not limited to,proteinaceous agents such as cytokines, peptide mimetics, and antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab or F(ab)₂ fragments or epitope binding fragments), nucleic acidmolecules (e.g., antisense nucleic acid molecules, RNAi and triplehelices), small molecules, organic compounds, and inorganic compounds.In particular, immunomodulatory agents include, but are not limited to,methothrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506(tacrolimus)), methylprednisolone (MP), corticosteroids, steriods,mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators. Examples ofimmunosupressant, include, but are not limited to, mycophenolate mofetil(CELLCEPT™), D-penicillamine (CUPRIMINE™, DEPEN™), methotrexate(RHEUMATREX™, TREXALL™), and hydroxychloroquine sulfate (PLAQUENIL™).

Immunomodulatory agents would also include substances that suppresscytokine production, downregulate or suppress self-antigen expression,or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see, U.S. Pat. No. 4,665,077),azathioprine (or cyclophosphamide, if there is an adverse reaction toazathioprine); bromocryptine; glutaraldehyde (which masks the MHCantigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypicantibodies for MHC antigens and MHC fragments; cyclosporin A; steroidssuch as glucocorticosteroids, e.g., prednisone, methylprednisolone, anddexamethasone; cytokine or cytokine receptor antagonists includinganti-interferon-gamma, -beta, or -alpha antibodies; anti-tumor necrosisfactor-alpha antibodies; anti-tumor necrosis factor-beta antibodies;anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; solublepeptide containing a LFA-3 binding domain (WO 90/08187 published Jul.26, 1990); streptokinase; TGF-.beta.; streptodomase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S.Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science251:430-432 (1991); WO 90/11294; and WO 91/01133); and T-Cell receptorantibodies (EP 340,109) such as T10B9.

Examples of cytokines include, but are not limited to lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormone such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-alpha; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoiotin (TPO); nerve growth factors suchas NGF-alpha; platelet-growth factor; transforming growth factors (TGFs)such as TGF-alpha and TGF-alpha; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CgP (GM-CSP); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such asTNF-alpha or TNF-beta; and other polypeptide factors including LIF andkit ligand (KL). As used herein, the term cytokine includes proteinsfrom natural sources or from recombinant cell culture and biologicallyactive equivalents of the native sequence cytokines. In certainembodiments, the methods further include administering to the subjectone or more immunomodulatory agents, preferably a cytokine. Preferredcytokines are selected from the group consisting of interleukin-1(IL-1), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF, thrombopoietin,and gamma interferon.

In certain embodiments, the immunomodulatory agent is a cytokinereceptor modulator. Examples of cytokine receptor modulators include,but are not limited to, soluble cytokine receptors (e.g., theextracellular domain of a TNF-alpha receptor or a fragment thereof, theextracellular domain of an IL-1beta receptor or a fragment thereof, andthe extracellular domain of an IL-6 receptor or a fragment thereof),cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-alpha,TNF-beta, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and GM-CSF),anti-cytokine receptor antibodies (e.g., anti-IL-2 receptor antibodies,anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN receptor antibodies, anti-TNF-alphaantibodies, anti-IL-1beta antibodies, anti-IL-6 antibodies, anti-IL-9,anti-IL-17 antibodies, antibodies, and anti-IL-12 antibodies). In aspecific embodiment, a cytokine receptor modulator is IL-4, IL-10, or afragment thereof. In another embodiment, a cytokine receptor modulatoris an anti-IL-1beta antibody, anti-IL-6 antibody, anti-IL-12 receptorantibody, anti-TNF-alpha antibody. In another embodiment, a cytokinereceptor modulator is the extracellular domain of a TNF-alpha receptoror a fragment thereof. In certain embodiments, a cytokine receptormodulator is not a TNF-alpha antagonist.

In certain embodiments, the immunomodulatory agent is a T cell receptormodulator. Examples of T cell receptor modulators include, but are notlimited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies(e.g., cM-T412 (Boeringer), IDEC-CE9.1 (IDEC and SKB), mAB 4162W94,Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies, anti-CD5antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40ligand monoclonal antibodies, anti-CD52 antibodies (e.g., CAMPATH 1H(Ilex)), anti-CD2 monoclonal antibodies) and CTLA4-immunoglobulin.

In certain embodiments, the immunomodulatory agent is a TNF-alphaantagonist. Examples of TNF-alpha antagonists include, but are notlimited to, antibodies (e.g., infliximab (REMICADE™; Centocor), D2E7(Abbott Laboratories/Knoll Pharmaceuticals Co., Mt. Olive, N.J.), CDP571which is also known as HUMIRA™ and CDP-870 (both of Celltech/Pharmacia,Slough, U.K.), and TN3-19.12 (Williams et al., 1994, Proc. Natl. Acad.Sci. USA 91: 2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci.USA 89:7375-7379)) soluble TNF-alpha receptors (e.g., sTNF-R1 (Amgen),etanercept (ENBREL™; Immunex) and its rat homolog RENBREL™, solubleinhibitors of TNF-alpha derived from TNFrI, TNFrII (Kohno et al., 1990,Proc. Natl. Acad. Sci. USA, 87:8331-8335), and TNF-alpha Inh (Seckingeret al, 1990, Proc. Natl. Acad. Sci. USA, 87:5188-5192)), IL-10, TNFR-IgG(Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA, 88:10535-10539),the murine product TBP-1 (Serono/Yeda), the vaccine CytoTAb(Protherics), antisense molecule 104838 (ISIS), the peptide RDP-58(SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC-333 (Dupont),VX-745 (Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco),NPI-13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix/AHP),CLX-120500 (Calyx), Thiazolopyrim (Dynavax), auranofin (Ridaura)(SmithKline Beecham Pharmaceuticals), quinacrine (mepacrinedichlorohydrate), tenidap (Enablex), Melanin (Large Scale Biological),and anti-p38 MAPK agents by Uriach.

An anti-ICOS immunotherapy may also be in conjunction with animmunoregulatory agent. In this approach, a chimeric, human or humanizedanti-ICOS antibody can be used. The term “immunoregulatory agent” asused herein for combination therapy refers to substances that act tosuppress, mask, or enhance the immune system of the host. This wouldinclude substances that suppress cytokine production, downregulate orsuppress self-antigen expression, or mask the MHC antigens. Examples ofsuch agents include 2-amino-6-aryl-5-substituted pyrimidines (see, U.S.Pat. No. 4,665,077), azathioprine (or cyclophosphamide, if there is anadverse reaction to azathioprine); bromocryptine; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone,methylprednisolone, and dexamethasone; cytokine or cytokine receptorantagonists including anti-interferon-γ, -β, or -α antibodies;anti-tumor necrosis factor-α antibodies; anti-tumor necrosis factor-βantibodies; anti-interleukin-2 antibodies and anti-IL-2 receptorantibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin;pan-T antibodies, for example anti-CD3 or anti-CD4/CD4a antibodies;soluble peptide containing a LFA-3 binding domain (WO 90/08187 publishedJul. 26, 1990); streptokinase; TGF-β; streptodomase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (U.S.Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science251:430-432 (1991); WO 90/11294; and WO 91/01133); and T-cell receptorantibodies (EP 340,109) such as T10B9. Examples of cytokines include,but are not limited to lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoiotin (TPO); nerve growth factors such as NGF-α;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-α; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CgP (GM-CSP); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 I, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines. Incertain embodiments, the methods further include administering to thesubject one or more immunomodulatory agents, for example a cytokine.Suitable cytokines may be selected from the group consisting ofinterleukin-1 (IL-1), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF,thrombopoietin, and γ interferon.

These immunoregulatory agents are administered at the same time or atseparate times from anti-ICOS antibodies. The preferred immunoregulatoryagent will depend on many factors, including the type of disorder beingtreated, as well as the patient's history, but the agent frequently maybe selected from cyclosporin A, a glucocorticosteroid (for exampleprednisone or methylprednisolone), azathioprine, bromocryptine,heterologous anti-lymphocyte globulin, or a mixture thereof.

5.30. Combination with Other Therapeutic Agents

Agents that act on the tumor neovasculature can also be used inconjunction with anti-ICOS immunotherapy and include tubulin-bindingagents such as combrestatin A4 (Griggs et al., Lancet Oncol. 2:82,(2001)) and angiostatin and endostatin (reviewed in Rosen, Oncologist5:20 (2000), incorporated by reference herein). Immunomodulatorssuitable for use in combination with anti-ICOS antibodies include, butare not limited to, of α-interferon, γ-interferon, and tumor necrosisfactor alpha (TNFα). In certain embodiments, the therapeutic agents usedin combination therapies using compositions and methods of the inventionare peptides.

In certain embodiments, an anti-ICOS immunotherapy is in conjunctionwith one or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ1^(I), γ2^(I),γ3^(I), N-acetyl-γ1^(I), PSAG and 011 Hinman et al., Cancer Research53:3336-3342 (1993) and Lode et al., Cancer Research 58: 2925-2928(1998)).

In certain embodiments, a treatment regimen includes compounds thatmitigate the cytotoxic effects of an anti-ICOS antibody composition.Such compounds include analgesics (e.g., acetaminophen),bisphosphonates, antihistamines (e.g., chlorpheniramine maleate), andsteroids (e.g., dexamethasone, retinoids, deltoids, betamethasone,cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids,mineralocorticoids, estrogen, testosterone, progestins).

In certain embodiments, the therapeutic agent used in combination withan anti-ICOS immunotherapy is a small molecule (i.e., inorganic ororganic compounds having a molecular weight of less than about 2500daltons). For example, libraries of small molecules may be commerciallyobtained from Specs and BioSpecs B.V. (Rijswijk, The Netherlands),Chembridge Corporation (San Diego, Calif.), Comgenex USA Inc.(Princeton, N.J.), and Maybridge Chemicals Ltd. (Cornwall PL34 OHW,United Kingdom).

In certain embodiments an anti-ICOS immunotherapy can be administered incombination with an anti-bacterial agent. Non-limiting examples ofanti-bacterial agents include proteins, polypeptides, peptides, fusionproteins, antibodies, nucleic acid molecules, organic molecules,inorganic molecules, and small molecules that inhibit and/or reduce abacterial infection, inhibit and/or reduce the replication of bacteria,or inhibit and/or reduce the spread of bacteria to other cells orsubjects. Specific examples of anti-bacterial agents include, but arenot limited to, antibiotics such as penicillin, cephalosporin, imipenem,axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol,erythromycin, clindamycin, tetracycline, streptomycin, tobramycin,gentamicin, amikacin, kanamycin, neomycin, spectinomycin, trimethoprim,norfloxacin, rifampin, polymyxin, amphotericin B, nystatin,ketocanazole, isoniazid, metronidazole, and pentamidine.

In certain embodiments an anti-ICOS immunotherapy can be administered incombination with an anti-fungal agent. Specific examples of anti-fungalagents include, but are not limited to, azole drugs (e.g., miconazole,ketoconazole (NIZORAL®), caspofungin acetate (CANCIDAS®), imidazole,triazoles (e.g., fluconazole (DIFLUCAN®)), and itraconazole(SPORANOX®)), polyene (e.g., nystatin, amphotericin B (FUNGIZONE®),amphotericin B lipid complex (“ABLC”) (ABELCET®), amphotericin Bcolloidal dispersion (“ABCD”) (AMPHOTEC®), liposomal amphotericin B(AMBISONE®)), potassium iodide (KI), pyrimidine (e.g., flucytosine(ANCOBON®), and voriconazole (VFEND®)). Administration of anti bacterialand anti-fungal agents can mitigate the effects or escalation ofinfectious disease that may occur in methods of the invention where apatient's ICOS expressing T cells are significantly depleted.

In certain embodiments of the invention, an anti-ICOS immunotherapy canbe administered in combination with one or more of the agents describedabove to mitigate the toxic side effects that may accompanyadministration of compositions of the invention. In other embodiments,an anti-ICOS immunotherapy can be administered in combination with oneor more agents that are well known in the art for use in mitigating theside effects of antibody administration, chemotherapy, toxins, or drugs.

In embodiments of the invention where an anti-ICOS immunotherapy isadministered in combination with another antibody or antibodies and/oragent, the additional antibody or antibodies and/or agents can beadministered in any sequence relative to the administration of theantibody of this invention. For example, the additional antibody orantibodies can be administered before, concurrently with, and/orsubsequent to administration of an anti-ICOS antibody or immunoconjugateto the human subject. The additional antibody or antibodies can bepresent in the same pharmaceutical composition as an antibody of theinvention, and/or present in a different pharmaceutical composition. Thedose and mode of administration of an antibody of this invention and thedose of the additional antibody or antibodies can be the same ordifferent, in accordance with any of the teachings of dosage amounts andmodes of administration as provided in this application and as are wellknown in the art.

5.31. Use of Anti-ICOS Antibodies in Diagnosing T Cell Malignancies

The present invention also encompasses anti-ICOS antibodies, andcompositions thereof, that immunospecifically bind to the human ICOSantigen, which anti-ICOS antibodies are conjugated to a diagnostic ordetectable agent. In certain embodiments, the antibodies are anti-ICOSantibodies with enhanced effector function. Such anti-ICOS antibodiescan be useful for monitoring or prognosing the development orprogression of a T cell malignancy as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can be accomplished by coupling ananti-ICOS antibody that immunospecifically binds to the human ICOSantigen to a detectable substance including, but not limited to, variousenzymes, such as but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes. Anydetectable label that can be readily measured can be conjugated to ananti-ICOS antibody and used in diagnosing T cell malignancies. Thedetectable substance may be coupled or conjugated either directly to anantibody or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, e.g.,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as a diagnostics according to the present invention.In certain embodiments, the invention provides for diagnostic kitscomprising an anti-ICOS antibody conjugated to a diagnostic ordetectable agent.

5.32. Use of Anti-ICOS Antibodies in Monitoring Immune Reconstitution

The present invention also encompasses anti-ICOS antibodies, andcompositions thereof, that immunospecifically bind to the human ICOSantigen, which anti-ICOS antibodies are conjugated to a diagnostic ordetectable agent. Such anti-ICOS antibodies can be useful for monitoringimmune system reconstitution following immunosuppressive therapy or bonemarrow transplantation. Such monitoring can be accomplished by couplingan anti-ICOS antibody that immunospecifically binds to the human ICOSantigen to a detectable substance including, but not limited to, variousenzymes, such as, but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as, but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as, but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, but not limited to, luminol;bioluminescent materials, such as, but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as, but not limitedto, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), and technetium(⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, 105Rh,⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ³⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,and ¹¹⁷Tin; positron-emitting metals using various positron-emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes. Anydetectable label that can be readily measured can be conjugated to ananti-ICOS antibody and used in diagnosing an autoimmune disease ordisorder. The detectable substance may be coupled or conjugated eitherdirectly to an antibody or indirectly, through an intermediate (such as,for example, a linker known in the art) using techniques known in theart. See, e.g., U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as a diagnostics according to thepresent invention. In certain embodiments, the invention provides fordiagnostic kits comprising an anti-ICOS antibody conjugated to adiagnostic or detectable agent.

5.33. Use of Anti-ICOS Antibodies in Diagnosing Autoimmune Diseases orDisorders

The present invention also encompasses anti-ICOS antibodies, andcompositions thereof, that immunospecifically bind to the human ICOSantigen, which anti-ICOS antibodies are conjugated to a diagnostic ordetectable agent. In certain embodiments, the antibodies are anti-ICOSantibodies with enhanced effector function. Such anti-ICOS antibodiescan be useful for monitoring or prognosing the development orprogression of an autoimmune disease or disorder as part of a clinicaltesting procedure, such as determining the efficacy of a particulartherapy. Such diagnosis and detection can be accomplished by coupling ananti-ICOS antibody that immunospecifically binds to the human ICOSantigen to a detectable substance including, but not limited to, variousenzymes, such as but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,), carbon (¹⁴C), sulfur (³⁵S), tritium(3H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, 90Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin;positron emitting metals using various positron emission tomographies,noradioactive paramagnetic metal ions, and molecules that areradiolabelled or conjugated to specific radioisotopes. Any detectablelabel that can be readily measured can be conjugated to an anti-ICOSantibody and used in diagnosing an autoimmune disease or disorder. Thedetectable substance may be coupled or conjugated either directly to anantibody or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, e.g.,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as a diagnostics according to the present invention.In certain embodiments, the invention provides for diagnostic kitscomprising an anti-ICOS antibody conjugated to a diagnostic ordetectable agent.

5.34. KITS

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with a composition of the invention for theprevention, treatment, management or amelioration of a T cell-mediateddisease and disorder, such as, but not limited to, chronic infection,autoimmune disease or disorder, inflammatory disease or disorder,graft-versus-host disease (GVHD), transplant rejection, and T cellproliferative disorder, or one or more symptoms thereof, potentiated byor potentiating a T cell-mediated disease and disorder.

The present invention provides kits that can be used in theabove-described methods. In one embodiment, a kit comprises acomposition of the invention, in one or more containers. In anotherembodiment, a kit comprises a composition of the invention, in one ormore containers, and one or more other prophylactic or therapeuticagents useful for the prevention, management or treatment of a Tcell-mediated disease and disorder, or one or more symptoms thereof,potentiated by or potentiating a T cell-mediated disease and disorder inone or more other containers. The kit may further comprise instructionsfor preventing, treating, managing or ameliorating a T cell-mediateddisease and disorder, as well as side effects and dosage information formethod of administration. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

6. SPECIFIC EMBODIMENTS

1. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand a variant Fc region, wherein the antibody mediates enhanced ADCCactivity as compared to the level of ADCC activity mediated by a parentantibody comprising the VH and VK domains and a wild type Fc region.

2. The antibody of embodiment 1, wherein the EC50 of the antibody asmeasured in an in vitro ADCC assay is at least about 7× lower than theEC50 value of the parent antibody.

3. The antibody of embodiments 1 or 2, wherein the variant Fc region hasa higher affinity for an Fc receptor than the wild type Fc region.

4. The antibody of embodiment 3, wherein the Fc receptor is humanFcgammaRIIIA.

5. The antibody of embodiment 1, wherein the variant Fc region comprisesat least one substitution of an amino acid residue selected from thegroup consisting of: residue 239, 330, and 332, wherein the amino acidresidue positions are determined according to the EU convention.

6. The antibody of embodiment 1, wherein the variant Fc region comprisesat least on amino acid substitution selected from the group consistingof: S239D, A330L, and 1332E; wherein the amino acid residue positionsare determined according to the EU convention.

7. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand an engineered Fc region, wherein the antibody has complexN-glycoside-linked sugar chains bound to the engineered Fc region inwhich fucose is not bound to N-acetylglucosamine in the reducing end inthe sugar chain.

8. The antibody of embodiment 7, wherein the antibody mediates enhancedADCC activity as compared to the level of ADCC activity mediated by aparent antibody comprising the VH and VK domains and a non-engineered Fcregion.

9. The antibody of embodiment 8, wherein the EC50 of the antibody asmeasured in an in vitro ADCC assay is at least about 7× lower than theEC50 value of the parent antibody.

10. The antibody of any one of the embodiments 1-7, wherein the VHdomain comprises the amino acid sequence of SEQ ID NO:7 and the VKdomain comprises the amino acid sequence of SEQ ID NO:2.

11. A nucleic acid encoding the amino acid sequence of the antibody asin any one of the embodiments 1-10.

12. The nucleic acid of embodiment 11, wherein the nucleic acidcomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:28-31.

13. A vector comprising the nucleic acid of embodiment 11.

14. The vector of embodiment 13, wherein the vector comprises anucleotide sequence selected from the group consisting of SEQ IDNO:28-31.

15. An isolated cell comprising the vector of embodiment 13.

16. The isolated cell of embodiment 15, wherein said cell lacks theactivity of a glycosylation enzyme.

17. The glycosylation enzyme of embodiment 16, wherein said enzyme isselected from the group consisting of FUT8 or GnTIII.

18. The isolated cell of embodiment 16, wherein the enzyme is selectedfrom the group consisting of FUT8 or GnTIII, and wherein the cellcomprises a vector comprising the nucleotide sequence selected from thegroup consisting of SEQ ID NO:28-31.

19. An isolated cell expressing the antibody as in any one of theembodiments 1-10.

20. A method of producing an antibody comprising culturing the isolatedcell of embodiment 19 under conditions sufficient for the production ofthe antibody and recovering the antibody from the culture.

21. A pharmaceutical composition comprising the antibody as in any oneof the embodiments 1-10 in a pharmaceutically-acceptable carrier.

22. The pharmaceutical composition of embodiment 21, wherein theantibody is of the IgG1, IgG2, IgG3, or IgG4 human isotype.

23. A method of treating an autoimmune disease or disorder in a human,comprising administering to a human in need thereof atherapeutically-effective amount of the antibody as in any one of theembodiments 1-10.

24. The method of embodiment 23, wherein the autoimmune disease ordisorder is SLE or scleroderma.

25. A method of treating or preventing rejection in a human transplantpatient, comprising administering to a human in need thereof atherapeutically-effective amount of the antibody as in any one of theembodiments 1-10.

26. A method of treating a T cell malignancy in a human comprisingadministering to a human in need thereof a therapeutically-effectiveamount of the antibody as in any one of the embodiments 1-10.

27. A method of treating an inflammatory disease or disorder in a human,comprising administering to a human in need thereof atherapeutically-effective amount of the antibody as in any one of theembodiments 1-10.

28. The method of embodiment 27, wherein the inflammatory disease ordisorder is myositis.

29. The method of embodiment 28, wherein the myositis is inclusion-bodymyositis (IBM), polymyositis (PM) or dermatomyositis (DM).

30. A method of depleting ICOS expressing T cells in a human patientcomprising administering to a human in need thereof atherapeutically-effective amount of the antibody as in any one of theembodiments 1-10.

31. The method of embodiment 30, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

32. The method of embodiment 30, wherein at least about 95% of the Tcells are depleted.

33. The method of embodiment 30, wherein the ICOS expressing T cell is amemory T cell.

34. The method of embodiment 30, wherein the ICOS expressing T cell is acirculating T cell.

35. A method of disrupting germinal center architecture in a secondarylymphoid organ of a primate, comprising administering an effectiveamount of the antibody as in any one of the embodiments 1-10.

36. The method of embodiment 35, wherein the primate is a non-humanprimate.

37. A method of depleting germinal center B cells from a secondarylymphoid organ of a primate comprising administering an effective amountof the antibody as in any one of the embodiments 1-10.

38. The method of embodiment 37, wherein the primate is a non-humanprimate.

39. The method of embodiment 37, wherein the primate is a human.

40. The method of embodiment 37, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

41. A method of depleting circulating class switched B cells in aprimate comprising administering an effective amount of the antibody asin any one of the embodiments 1-10.

42. The method of embodiment 41, wherein the primate is a non-humanprimate.

43. The method of embodiment 41, wherein the primate is a human.

44. The method of embodiment 41, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

45. The method of embodiment 41, wherein at least about 95% of thecirculating class switched B cells are depleted.

46. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand a variant Fc region, wherein the antibody mediates enhanced ADCCactivity as compared to the level of ADCC activity mediated by a parentantibody comprising the VH and VK domains and a wild type Fc region, andwherein said antibody is capable of depleting germinal center B cellsfrom a secondary lymphoid organ of a primate.

47. The antibody of embodiment 46, wherein the primate is a non-humanprimate.

48. The antibody of embodiment 46, wherein the primate is a human.

49. The antibody of embodiment 46, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

50. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand a variant Fc region, wherein the antibody mediates enhanced ADCCactivity as compared to the level of ADCC activity mediated by a parentantibody comprising the VH and VK domains and a wild type Fc region, andwherein said antibody is capable of depleting circulating class switchedB cells in a primate.

51. The antibody of embodiment 50, wherein the primate is a non-humanprimate.

52. The antibody of embodiment 50, wherein the primate is a human.

53. The antibody of embodiment 50, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

4. The antibody of embodiment 50, wherein at least about 95% of thecirculating class switched B cells are depleted.

5. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand an engineered Fc region, wherein the antibody has complexN-glycoside-linked sugar chains bound to the engineered Fc region inwhich fucose is not bound to N-acetylglucosamine in the reducing end inthe sugar chain, wherein the antibody mediates enhanced ADCC activity ascompared to the level of ADCC activity mediated by a parent antibodycomprising the VH and VK domains and a non-engineered Fc region, andwherein said antibody is capable of depleting germinal center B cellsfrom a secondary lymphoid organ of a primate.

56. The antibody of embodiment 55, wherein the primate is a non-humanprimate.

57. The antibody of embodiment 55, wherein the primate is a human.

58. The antibody of embodiment 55, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

59. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand an engineered Fc region, wherein the antibody has complexN-glycoside-linked sugar chains bound to the engineered Fc region inwhich fucose is not bound to N-acetylglucosamine in the reducing end inthe sugar chain, wherein the antibody mediates enhanced ADCC activity ascompared to the level of ADCC activity mediated by a parent antibodycomprising the VH and VK domains and a non-engineered Fc region, andwherein said antibody is capable of depleting class switched B cells ina primate.

60. The antibody of embodiment 59, wherein the primate is a non-humanprimate.

61. The antibody of embodiment 59, wherein the primate is a human.

62. The antibody of embodiment 59, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody.

63. The antibody of embodiment 59, wherein at least about 95% of thecirculating class switched B cells are depleted.

7. EXAMPLES 7.1. Construction of ADCC Enhanced Anti-ICOS Antibodies

The following sections describe the design of an ADCC enhanced anti-ICOSantibody comprising a human IgHγ1 constant region. An ADCC enhancedanti-ICOS antibody may comprise variant Fc regions with increasedeffector function (see, US Patent Publication No's: US 2007-0003546 A1,US20060160996A9, US 2005-0054832 A1, US 2004-0132101 A1, and US2004-0110226 A1). An ADCC enhanced anti-ICOS antibody may comprisecomplex N-glycoside-linked sugar chains linked to Asn297 of the Fcregion in which fucose is not bound to N-acetylglucosamine in thereducing end (see, U.S. Pat. No. 6,946,292, US Patent Publication No's:US 2006-0223147 A1, US 2006-0021071 A1, US 2005-0272916 A1, US2004-0259150 A1, US 2004-0132140 A1, US 2004-0110704 A1, and US2004-0110282 A1). The ADCC enhanced anti-ICOS antibodies described inthe Examples comprise the heavy and light chain variable domains of theJMab-136 anti-ICOS antibody described in U.S. Pat. No. 6,803,039. Theamino acid sequence of the JMab anti-ICOS antibody VH and VK domains aredisclosed herein as SEQ ID NO: 7 and 2, respectively. The anti ICOSantibody comprising JMab-136 VH and VL domains and further comprisingthe IgHγ2 constant region is hereinafter referred to as IC009. The antiICOS antibody comprising JMab-136 VH and VL domains and furthercomprising the IgHγ1 constant region is hereinafter referred to as asIC9G1. Those skilled in the art recognize that the experimental methodsdescribed herein may also be applied to any other anti-ICOS antibody,for example, but not limited to, those described in U.S. Pat. No.6,803,039.

7.1.1. Sequence Optimization

Amino acid sequence: The amino acid sequence of the VK domain (SEQ IDNO: 1) comprises the following motifs: a potential o-glycosylation siteat amino acid position 5, and a potential deamidation motif at aminoacid position 92 in the VK CDR3. The amino acid sequence of the VHdomain (SEQ ID NO: 6) comprises the following motifs: a potentialo-glycosylation site at amino acid position 17, and a potentialisoaspartate formation motif at amino acid position 99 in the VH CDR3.Amino acid positions are determined according to the Kabat consensus.The amino acid sequence of the VH or VK domain may be changed toeliminate any one of these sequence motifs and thus eliminate thepotential for posttranslational modification at the altered sequencemotifs. For example, an NG potential deamidation motif may be eliminatedby substituting the N residue with a Y, D or G residue. Methods forintroducing a substitution into the amino acid sequence of the anti-ICOSantibody are described below. The antigen binding properties of an aminoacid substitution comprising anti-ICOS antibody may be ascertained usingthe methods described herein.

Nucleic acid sequence: The polynucleotides encoding the heavy and lightchains of the anti-ICOS antibody may be subjected to nucleic acidsequence optimization. The final goal of the sequence optimizationprocess is to create a coding region that is transcribed and translatedat the highest possible efficiency. Sequence optimization is achieved bya combination of: (i) codon usage optimization, (ii) G/C contentadaptation, (iii) elimination of internal splicing sites and prematurepolyadenylation sites, (iv) disruption of stable RNA secondarystructures, (v) elimination of direct repeat sequences, (vi) eliminationof sequences that may form stable dsRNA with host cell transcripts,(vii) eliminate sequences targeted by host cell micro RNAs, and (viii)introduction of RNA stabilizing and RNA translocation signals. Detailedsequence optimization methods are described in WO2004059556A2,WO2006015789A2, Bradel-Tretheway et al., J. Virol. Methods 111: 145-56(2003), Valencik & McDonald, Transgenic Res. 3:269-75 (2001).Alternatively, a sequence may be optimized by a commercial provider(e.g., GENEART Inc.).

Nucleotide sequences encoding the VH, VK, heavy chain and light chain ofthe IC9G1 were optimized following the methods described herein. Theoptimized nucleotide sequences encoding the VH, VK, heavy chain andlight chain of IC9G1 is disclosed as SEQ ID NO:28-31, respectively.

7.1.2. Gene Assembly and Expression Cloning

Constructs may be generated by a PCR-based gene assembly method firstdescribed by Stemmer (Stemmer, W. P. et al. 1995 Gene, 164:49-53). Thismethod consists of four steps: oligonucleotide synthesis; gene assembly;gene amplification and cloning. Eight VH gene specific primers and sixVK gene specific primers that may be used for PCR mediated gene assemblyare listed in Table 2. Primer sets for variant VH and VK regionscomprising specific amino acid substitutions may be generated bymodifying the nucleic acid sequence of the primer encoding the givenamino acid residue. Primers are designed to overlap by 15-20 nucleotidesand are ligated into a complete variable region during thermal cycling.In case of VH, an additional vector specific primer (Universal VH FW inTable 2.) is included in the PCR mediated gene assembly process. Theexternal 5′ and 3′ primers for VH region incorporate a uniquerecognition site for the XbaI and ApaI restriction endonuclease,respectively, to help with the subsequent cloning steps. The external 5′and 3′ primers for VK incorporate a unique recognition site for the XmaIand BsiWI restriction endonuclease, respectively, to help with thesubsequent cloning steps. PCR products of the correct size arerestriction digested and ligated in frame into an expression vectorwherein VH regions are digested with XbaI and ApaI, and VK regions aredigested with XmaI and BsiWI according to the manufacturer'sinstructions. The heavy chain assembly vector comprises eukaryotictranscription control elements operably linked to a polynucleotideencoding the MGDNDIHFAFLSTGVHS VH leader (SEQ ID NO: 26) and a humanIgHγ1 constant region wherein said transcription control elementscomprise a CMV immediate early promoter and a SV40 poly A additionsignal. The use of appropriately designed primers for VH assemblyensures that the polynucleotide sequences encoding the VH leader, VHregion and IgHγ1 constant region are joined in frame within the finalheavy chain expression vector. The light chain assembly vector compriseseukaryotic transcription control elements operably linked to apolynucleotide encoding the human VK1-L12 leader (amino acid sequenceMDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO:27); Bentley, D. L. & Rabbitts, T. H.,Nature 288, 730-733 (1980)) and a human IgLK constant region whereinsaid transcription control elements comprise a CMV immediate earlypromoter and a SV40 poly A addition signal. The use of appropriatelydesigned primers for VK assembly ensures that the polynucleotidesequences encoding the VK1-L12 leader, VK region and IgLK constantregion are joined in frame within the final light chain expressionvector. The ligation product is used to transform DH10B competent E.coli cells according to the manufacturer's protocols. Coloniescontaining the plasmid and a correct sized insert can be identifiedusing various methods known in the art (e.g. restriction digest ofvector DNA preparation, PCR amplification of vector sequences). Plasmidclones with correct sized insert may be sequenced using dideoxysequencing reaction (e.g., BigDye® Terminator v3.0 Cycle SequencingReady Reaction Kit, ABI). Plasmid DNA is prepared from selected clonesusing the QIAGEN Mini and Maxi Plasmid Kit according to themanufacturer's protocols.

DNA plasmid expression vector preparations encoding the anti-ICOS heavychain and light chain polypeptides are used to co-transfect HEK293cells. The co-transfected HEK293 cells are cultured under standardconditions. Antibody-containing conditioned medium is harvested 72 and144 hours post-transfection. The secreted, soluble human IgG is purifiedfrom the conditioned media directly using 1 ml HiTrap protein A columnsaccording to the manufacturer's instructions (APBiotech, Inc.,Piscataway, N.J.). Purified human IgG (typically >95% pure, as judged bySDS-PAGE) is dialyzed against phosphate buffered saline (PBS), flashfrozen and stored at −70° C.

IgG concentration of the purified preparation is quantified using acapture ELISA assay. Briefly, IgG molecules are captured on a 96-wellplate via an immobilized goat anti-human IgG H+L specific antibody, anddetected with an HRP conjugated anti-human kappa light chain antibody.The assay is calibrated using a reference IgG1 mAb of irrelevantspecificity.

TABLE 2 Representative primer sets for VH and VK regionassembly. Gene specific nucleotides are printedin upper case, vector specific nucleotides areprinted in lower case. Recognition sites forrestriction endonucleases used for VH and VK fragment cloning are underlined. Univ VH FWtatatatatctagacatatatatgggtgacaatgacatc cactttgcctttctctcc(SEQ ID NO: 11) VH FW1 tccactttgcctttctctccacaggtgtccactccCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG GGGCCTCAGTG (SEQ ID NO: 12)VH RE2 CATATAGTAGCCGGTGAAGGTGTATCCAGAAGCCTTGCAGGAGACCTTCACTGAGGCCCCAGGCTTC (SEQ ID NO: 13) VH FW3CACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGG ACAAGGGCTTGAGTGGATGGGATGGATC(SEQ ID NO: 14) VH RE4 CTGCCCTGAAACTTCTGTGCATAGTTTGTGCCACCACTGTGAGGGTTGATCCATCCCATCCAC (SEQ ID NO: 15) VH FW5CAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACG TCCATCAGCACAGCCTACATGGAGCTGAG(SEQ ID NO: 16) VH RE6 GTCCTCGCACAGTAATACACGGCCGTGTCGTCGGATCTCAGCCTGCTCAGCTCCATGTAGGCTG (SEQ ID NO: 17) VH FW7GTATTACTGTGCGAGGACGTATTACTATGATAGTAGTGG TTATTACCATGATGCTTTTGATATCTG(SEQ ID NO: 18) VH RE8 tatatatagggcccttggtggaggcCTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGATATCAAAAGCATC (SEQ ID NO: 19) VK FW1tatatataccccggggccaaatgtGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGA G (SEQ ID NO: 20) VK RE2GATACCAGGCTAACAACCTGCTAATACCCTGACTCGCCC GACAAGTGATGGTGACTCTGTCTCCTACAGA(SEQ ID NO: 21) VK FW3 GTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGTTGCATCCAGTTTGCAAAGTG (SEQ ID NO: 22) VK RE4GTGAAATCTGTCCCAGATCCACTGCCGCTGAACCTTGAT GGGACCCCACTTTGCAAACTGGATG(SEQ ID NO: 23) VK FW5 CTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAG (SEQ ID NO: 24) VK RE6tatatatacgtacgTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCCACGGGAAACTGTTAGCCTGTTGACAATAG TAAG (SEQ ID NO: 25)

7.1.3. ADCC Enhanced Anti-ICOS Antibody Comprising a Variant FC Domain

An antibody expression vector encoding an ADCC enhanced anti-ICOSantibody having a variant Fc domain comprising the S239D, A330L, andI332E amino acid substitutions (hereinafter referred to as “IC9G1-3M”)may be generated using the methods described in US Patent Publications2004/0132101 and 2005/0054832, both to Lazar et al. Briefly, the abovedescribed antibody expression vector encoding the JMab136 VH and VLdomains is modified using a site directed mutagenesis kit (e.g.,QuickChange (Promega)) to introduce the necessary nucleotide residuesubstitutions into the polynucleotide sequence encoding the heavy chainconstant region to generate the IC₉G1-3M antibody expression vector.Purified IC9G1-3M antibody is generated by transfecting HEK239F cellswith the IC9G1-3M antibody expression vector. Transfected cells are fedat day3 and 6 and the antibody-containing conditioned medium isharvested at day 9. Antibody is purified from the conditioned mediumusing a pre-cast protein A column (GE Healthcare). Antibody is elutedfrom the column with low pH buffer, neutralized, and dialyzed againstPBS. The concentration of the purified antibody is calculated from thesolution's optical density at 280 nm.

7.1.4. ADCC Null Anti-ICOS Fc Variant Antibody

An antibody expression vector encoding an anti-ICOS antibody withreduced ADCC activity having an Fc region comprising the L234F, L235E,and P331 S amino acid substitutions (hereinafter referred to as“IC9G1-TM”) is generated using methods described in US 2004/0132101 andUS 2005/0054832, both to Lazar et al. Briefly, the above describedantibody expression vector encoding the JMab136 VH and VL domains ismodified using a site directed mutagenesis kit (e.g., QuickChange(Promega)) to introduce the necessary nucleotide residue substitutionsinto the polynucleotide sequence encoding the heavy chain constantregion to generate the IC9G1-TM antibody expression vector. PurifiedIC9G1-TM antibody is generated by transfecting HEK239F cells with theIC9G1-TM antibody expression vector. Transfected cells are fed at day3and 6 and the antibody-containing conditioned medium is harvested at day9. Antibody is purified from the conditioned medium using a pre-castprotein A column (GE Healthcare). Antibody is eluted from the columnwith low pH buffer, neutralized, and dialyzed against PBS. Theconcentration of the purified antibody is calculated from the solution'soptical density at 280 nm.

7.1.5. Afucosylated Anti-ICOS Antibody with Increased ADCC

An IC9G1 antibody composition (hereinafter referred to as IC9G1-aFuc)comprising a plurality of antibodies having complex N-glycoside-linkedsugar chains linked to Asn297 of the Fc region in which fucose is notbound to N-acetylglucosamine in the reducing end was prepared accordingto the methods set forth in U.S. Pat. No. 6,946,292 to Kanda et al.Briefly, fucosyltransferase knock-out CHO cells are transfected with aDNA plasmid expression vector preparation encoding the heavy and lightchains of JMab136. Transfected cells are fed at day3 and 6 and theantibody-containing conditioned medium is harvested at day 9. Antibodyis purified from the conditioned medium using a pre-cast protein Acolumn (GE Healthcare). Antibody is eluted from the column with low pHbuffer, neutralized, and dialyzed against PBS. The concentration of thepurified antibody is calculated from the solution's optical density at280 nm.

7.2. Binding Profile Characterization of ADCC Enhanced Anti-ICOSAntibodies

The binding profile of ADCC enhanced anti-ICOS antibodies may becharacterized by a number of methods known to one of skill in the arts.The antibodies may be characterized by using, for example, but notlimited to, cell based ELISA assays, ELISA assays using a recombinantICOS molecule as capture reagent, flow cytometry, Biacore analysis.

The ability of an ADCC enhanced anti-ICOS antibody to bind ICOS may beassessed by a cell based ICOS binding assay utilizing stabletransfectants cells expressing recombinant ICOS protein on their cellsurface as a capture agent. U.S. Pat. No. 6,803,039 describes an ICOStransgenic CHO cell line and an ICOS transgenic HPB-ALL cell line, eachof which may be used in a cell based ELISA assay. A cell based ELISA maybe performed by using any one of the methods known to one skilled in thearts. For example, HPB-ALL h-ICOS+ cells are cultured according tostandard protocols in RPMI 1640 medium containing L-glutamine andsupplemented with 10% Fetal Calf Serum. Individual wells of a 96 well Ubottom plate are seeded with 1×10e5 stable transfectants HPB-ALL hICOScells and incubated overnight. Cells are washed once with ELISA bufferprior to incubation on ice with various amounts of anti-ICOS antibodies.Binding reactions are performed in triplicates for each antibodyconcentration tested. Negative control wells using an isotype matchedantibody of irrelevant specificity should be included in the assay.Additional negative control wells seeded with non-transfected HPB-ALLcells may also be used to further demonstrate the binding specificitythe anti-ICOS antibody. Following incubation with the antibody, HPB-ALLhICOScells are washed three times with 200 micro liter of ELISA buffer.The amount of anti-ICOS antibodies bound to HPB-ALL hICOS cells may bedetected using a goat anti-human kappa antibody conjugated withhorseradish peroxidase following standard protocols. An ICOS specificantibody should give a dose dependent ELISA signal with the HPB-ALLhICOS cells but not with the parental HPB-ALL cells. The ELISA signal isexpected to reach a maximum at an antibody concentration where allavailable epitopes on the cell surface are bound.

Anti-ICOS antibodies may also be characterized by an ELISA assay thatuses a recombinant ICOS-Fc fusion protein (R&D Systems) as a capturereagent. ELISA assays may be performed according to any one of theestablished protocols known to one of skill in the art. For example,microtiter plates are coated with ICOS-Fc fusion protein (e.g., 100 μlof 0.25 μg/ml ICOS-Fc protein) and incubated at 4° C. overnight. Anyremaining binding sites are blocked with 4% skimmed milk in PBS buffer(blocking buffer) for 1 h at 37° C. Approximately 25-50 μl of anti-ICOSantibody solution of various concentrations is added to each well andincubated for 1 h at 37° C. After washing the wells, a goat anti-humankappa antibody conjugated with horseradish peroxidase is used for thedetection of ICOS-Fc fusion protein bound anti-ICOS antibody followingthe manufacturer's directions. Detection is carried out by adding 30 μlof tetramethylbenzidine (TMB) substrate (Pierce) followed byneutralization with 30 μl of 0.2 M H₂SO₄. The absorbance is read at 450nm. Negative control wells using an isotype matched antibody ofirrelevant specificity should be included in the assay. In addition,negative control wells without ICOS-Fc protein may also be included inthe assay. An ICOS specific antibody should give a dose dependent ELISAsignal with the ICOS-Fc coated wells but not with the uncoated negativecontrol wells. The ELISA signal is expected to reach a maximum at anantibody concentration where all available epitopes are occupied.

The antigen specificity of ADCC enhanced anti-ICOS antibodies may alsobe characterized by flow cytometry assays. Isolated cells expressinghuman ICOS on their cell surface (e.g., stable transfectant CHO hICOScells, activated T lymphocytes) are incubated with a fluorescentlyconjugated anti-ICOS antibody following a standard protocols. Negativecontrol cell that do not display ICOS on the cell surface are stained aswell using the same protocol. Immuno stained cells are analyzed on aflow cytometer. Cells incubated with a negative control antibody ofunrelated specificity may also be included in the assay. An ICOSexpressing cell stained with a fluorescently conjugated anti-ICOSantibody should have a mean fluorescence intensity that is higher thanthat of either a non-ICOS expressing cell stained with the same antibodyor an ICOS expressing cell stained with a negative control antibody ofirrelevant specificity.

The binding affinity of ADCC enhanced anti-ICOS antibodies may also bedetermined using the Biacore System (see, U.S. Pat. No. 6,803,039).

7.3. Antigen Binding Affinity of Deamidated Anti-ICOS Antibodies

Deamidation of asparagines residue may significantly contribute to thechemical degradation of antibody pharmaceuticals (see, Chelius et al.,Anal. Chem. 77:6004-11 (2005)). Deamidation may be especially importantwhen the potential deamidation site is located within the CDR regions ofan antibody. CDR3 of the JMAb 136 light chain variable domain comprisesa NS potential deamidation site at Kabat position 92. The effect ofdeamidation on the antigen binding affinity of an anti-ICOS antibody maybe assessed using methods known to one of skills in the art. Briefly, ananti-ICOS antibody is stored under conditions known to enhance thechemical deamidation process. For example, an anti-ICOS antibody may bestored for two weeks at 40° C. in a buffer with a pH of 8.5 or 9.5 toaccelerate the deamidation process. As deamidation of an asparagineresidue changes the overall charge of the protein, the extent ofdeamidation in a given purified antibody sample may be assessed by anumber of analytical methods, for example, but not limited to, ionexchange chromatography (IEC), isoelectro focusing (IEF), LiquidChromatography/Mass Sprectometry (LC-MS). The effect of deamidation onICOS binding affinity may be ascertained by comparing the bindingproperties of IC9G1 antibody preparations with high and low levels ofdeamidation. Binding affinity of the various antibody preparations maybe analaysed by for example, but not limited to, cell based ELISAassays, ELISA assays using a recombinant ICOS molecule as capturereagent, flow cytometry, Biacore analysis. A significant decrease in theICOS binding activity of a IC9G1 anti-ICOS antibody preparations upondeamidation would suggest that deamidation plays a major role in thechemical degradation of the antibody. Alternatively, the ICOS bindingactivity of non-deamidated and deamidated IC9G1 antibody preparationsmay be very similar suggesting that deamidation is not a major concernwhen considering the degradation pathways of the antibody. Ifdeamidation poses a problem for the long term stability of the IC9G1anti-ICOS antibody, then the deamidation site may be eliminated from theamino acid sequence by generating single amino acid substitutionvariants using the methods described above. The ICOS binding affinity ofany deamidation null anti-ICOS antibody variant may be characterized byusing the methods described herein.

7.4. In Vitro ADCC Activity of ADCC Enhanced Anti-ICOS Antibodies

The ADCC activity of various anti-ICOS antibodies may be determined byan in vitro ADCC assay, such as that described in U.S. Pat. No.5,500,362 or 5,821,337. The ADCC assay may be performed using acommercially available assay kit, for example, but not limited toCytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). The CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega) is a calorimetricalternative to ⁵¹Cr release cytotoxicity assays. The CytoTox 96® Assayquantitatively measures lactate dehydrogenase (LDH), a stable cytosolicenzyme that is released upon cell lysis. Released LDH in culturesupernatants is measured with a 30-minute coupled enzymatic assay, whichresults in the conversion of a tetrazolium salt (INT) into a redformazan product. The amount of color formed is proportional to thenumber of lysed cells.

The assays are performed according to the manufacturer's directions.Briefly, target cells are washed with PBS, resuspended in RPMI-5 PhenolFree media at a cell density of 0.4×10⁶/ml. NK effector cells are washedonce in PBS and resuspended in RPMI-5 Phenol Free media at a celldensity 1×10⁶/ml. Assays are performed in U bottom 96 well plates. Eachassay plate includes a combination of experimental and control wells.Experimental wells are set up by combining 50 μl of the appropriateantibody dilution, 50 ul of target cell suspension and 50 ul of effectorcell suspension. The cell densities described above result in a 1:2.5target to effector cell ratio; effector cell stock may be furtherdiluted or concentrated if a different target to effector ratio isdesired. Several different types of control wells are used to accountfor (i) the spontaneous LDH release form target cells (TargetSpontaneous), (ii) the spontaneous LDH release from effector cells(Effector Spontaneous), (iii) the maximum LDH release from the targetcells (Target Maximum), and (iv) the presence of contaminants in theculture medium (Background). All wells in use on a 96 well plate containthe same final volume. Reactions are set up in triplicates. Followingset up, plates are spun at 120×g for 3 minutes to pellet the cells.Incubate plate at 37° C./5% CO₂ for 4 hours. Forty five minutes prior tothe end of incubation 15 μl of manufacturer provided Lysis Buffer isadded to the Target Cell Maximum Release Control well. After incubationthe plate is centrifuged at 120×g for 4 minutes. 50 μl of thesupernatant from each well is transferred to a new flat bottom 96 wellplate. 50 μl of reconstituted substrate mix (assembled from manufacturerprovided components) is added and the plate is incubated at roomtemperature 10-20 minutes protected from light. 50 μl of manufacturerprovided stop buffer is added and absorbance at 490 or 492 nm ismeasured in a plate reader. % cytotoxicity equals (Experimental-Effectorspontaneous−Target Spontaneous)/(Target Maximum−Target Spontaneous).Prior to calculating the % cytotoxicity all other values are reduced bythe Background.

Potential target cells for an anti-ICOS antibody dependent cytotoxicityassay include, but are not limited to, stable transfectant hICOSexpressing cell lines (e.g., human ICOS expressing CHO cell line andhuman ICOS expressing HPB-ALL cell line described in U.S. Pat. No.6,803,039). Alternatively, freshly isolated cells displaying human ICOSon their cell surface (e.g., activated T cells) may also be used astarget cells. Suitable effector cells include, but are not limited to,freshly isolated natural killer cells (NK cells), and peripheral bloodmononuclear cells (PMBC). NK cell lines expressing a transgenic Fcreceptor (e.g. CD16) and associated signaling polypeptide (e.g. FCεRI-γ)may also serve as effector cells (see, e.g. WO 2006/023148 A2 toCampbell).

ADCC assays are performed in parallel using unmodified anti-ICOSantibody (e.g., IC9G1), ADCC enhanced anti-ICOS antibody (e.g.,IC9G1-aFuc, IC9G1-3M). ADCC enhanced antibodies are expected to mediatethe lysis of a higher percentage of target cells than that of mediatedby the unmodified antibody. An anti-ICOS antibody with reduced ADCCactivity (e.g., IC9G1-TM) may also be include in the assay as a negativecontrol. Target specificity of the anti-ICOS mediated ADCC assay may bedemonstrated by using target cells not expressing hICOS. The backgroundcytotxicity in ADCC assays performed using target cells not expressinghICOS is expected to be similar between reactions using ADCC enhancedand unmodified anti-ICOS antibodies.

7.5.Human ICOS Expression in Transgenic Mice

Human ICOS transgenic mice, which can be developed using methods wellknown to persons trained in the art, or other transgenic animalsexpressing human ICOS can be used to assess different therapeuticregimens comprising anti-ICOS antibodies, such as variations in dosingconcentration, amount, and timing. The efficacy in human patients ofdifferent therapeutic regimens can be predicted using, e.g., the twoindicators described below, i.e., T cell depletion in certain bodilyfluids and/or tissues and the ability of an anti-ICOS antibody to bind Tcells. In particular embodiments, treatment regimens that are effectivein human ICOS transgenic mice could be used with compositions andmethods of the invention to treat human T cell disorders and diseaseincluding, but not limited to, autoimmune diseases or disorders,inflammatory diseases or disorders, and T cell malignancies.

In order to determine whether human ICOS is expressed on T cellsubpopulations from transgenic mice (hICOStg) comprising the human ICOStransgene, T cells could be extracted from the thymus, peripheral blood,spleen, lymph node and peritoneal lavage of these mice. Human ICOS andmouse ICOS expression could be assessed in these cells by contacting thecells with anti-ICOS antibodies that specifically bind human ICOS (e.g.,IC9G1) or mouse ICOS (mICOS) (e.g., clone 15F9, BioLegend, CA). Bindingof the antibody to T lineage cell subpopulations could be detected usingfour-color immunofluorescence staining with flow cytometry analysis. Therelative expression levels of mICOS and hICOS, could then be assessed bymeasuring mean fluorescence intensity (anti-hICOS for hICOS andanti-mICOS for mICOS) respectively.

7.6. Anti-ICOS Antibody Mediated Depletion of T Cells In Vivo

Anti-ICOS antibodies of the invention, which bind to human ICOS, can beassessed for their ability to deplete hICOStg thymic, peripheral blood,splenic, and lymph node T cell subpopulations in vivo. For example, eachantibody would be given to mice at either 250 or 50 μg/mouse, a singledose about 10 to 50-fold lower than a 375 mg/m² dose given to a humansubject. T cell depletion from thymus, blood, spleen and lymph nodes ofhICOStg mice would be determined by immunofluorescence staining withflow cytometry analysis. The results using anti-ICOS antibodiesidentified as capable of depleting T cells can be correlated to use inhumans and antibodies with properties of the identified antibodies canbe used in the compositions and methods of the invention for thetreatment of human T cell disorders and disease including, but notlimited to, autoimmune diseases or disorders, inflammatory diseases ordisorders, and T cell malignancies.

7.6.1. Determination Whether Tissue T Cell Depletion is FCγR-Dependent

Should administration of an anti-ICOS mAb of the invention result intissue T cell depletion, the following assays can be used to demonstratedependence upon FcγR expression. Through a process of interbreedinghICOStg mice with mice lacking expression of certain FcγR, mice can begenerated that express hICOS and lack expression of certain FcγR. Suchmice can be used in assays to assess the ability of anti-ICOS antibodiesto deplete T cells through pathways that involve FcγR expression, e.g.,ADCC. Thus, anti-ICOS antibodies identified in these assays can be usedto engineer anti-ICOS antibodies with enhanced effector function usingthe techniques described above. Such antibodies can in turn be used inthe compositions and methods of the invention for the treatment of humanT cell disorders and disease including, but not limited to, autoimmunediseases or disorders, inflammatory diseases or disorders, and T cellmalignancies.

Mouse effector cells express four different FcγR classes for IgG, thehigh-affinity FcγRI (CD64), and the low-affinity FcγRII (CD32), FcγRIII(CD16), and FcγRIV molecules. FcγRI, FcγRIII and FcγRIV arehetero-oligomeric complexes in which the respective ligand-binding achains associate with a common γ chain (FcRγ). FcRγ chain expression isrequired for FcγR assembly and for FcγR triggering of effectorfunctions, including phagocytosis by macrophages. Since FcRγ^(−/−) micelack high-affinity FcγRI (CD64) and low-affinity FcγRIII (CD16) andFcγRIV molecules, FcRγ^(−/−) mice expressing hICOS can be used to assessthe role of FcγR in tissue T cell depletion following anti-ICOS antibodytreatment.

7.6.2. Durability of Anti-ICOS Antibody-Induced T cell Depletion

To assess the efficacy and duration of T cell depletion, hICOStg micecan be administered a single low dose (e.g. 250 μg) injection ofanti-ICOS antibody and the duration and dose response of T celldepletion followed as a function of time. The results are expected todemonstrate that circulating T cells are depleted for a substantialamount of time (e.g. one week to six months), followed by a gradualrecovery of ICOS expressing T cells.

7.7. Therapeutic Efficacy of Subcutaneous (S.C.) Administration of anAnti-ICOS Antibody of the Invention

The assay described herein can be used to determine whether asubcutaneous route of administration of an anti-ICOS antibody of theinvention can effectively deplete T cell subpopulations. The results ofthe efficacy of different delivery routes tested in animal models can becorrelated to humans by means well-known in the art.

For example, hICOStg mice can be treated with an anti-ICOS antibody ofthe invention at 250 μg either by subcutaneous (s.c.), intraperitoneal(i.p.) or intravenous (i.v.) administration. Values are determined forthe mean (±SEM) blood (per mL), thymus, spleen, lymph node, andperitoneal cavity ICOS positive T cell numbers on day seven as assessedusing flow cytometry. Results are expected to demonstrate thatsubcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.)administration of an anti-ICOS antibody of the invention willeffectively deplete ICOS expressing circulating and tissue T cells invivo.

7.8. Use of Anti-ICOS Antibodies in Reducing Tumor Growth in an In VivoLymphoma Model

Anti-ICOS antibodies of the invention, which bind to human ICOS, may beassessed for their ability to reduce tumor growth in in vivo animalmodels. For example, SCID mice would be injected with human ICOSexpressing cell lines to establish a tumor xenograft (e.g., stabletransfectant HBP-ALL hICOS cells). Subsequently, the mice would be givenseveral doses of an anti-ICOS antibody of the invention (e.g., 100 μgantibody/mouse 5 times). Tumor growth would be followed using standardmethods (e.g., tumor volume, animal weight, paralysis) The effect ofanti-ICOS treatment on tumor growth may be determined by comparinganimals receiving anti-ICOS or control antibody treatment. The resultsobtained using anti-ICOS antibodies identified as capable of reducingtumor growth can be correlated to use in humans, and antibodies capableof reducing tumor growth can be used in the compositions and methods ofthe invention for the treatment of human T cell disorders and diseasesincluding, but not limited to, autoimmune diseases or disorders,inflammatory diseases or disorders, and T cell malignancies.

To determine whether an anti-ICOS antibody's ability to reduce tumorgrowth is dependent on ICOS density, tumor cell lines with differentICOS expression profiles may be tested in the above described in vivotumor growth assay. The results obtained may demonstrate whether humanICOS density on the tumor cell surface can influence the tumor growthreducing activity of an anti-ICOS antibody. The results can becorrelated to treatment of human patients with varying levels of ICOSexpression. Thus, the methods for examining ICOS presence and density,described herein, can be used in human subjects to identify patients orpatient populations for which certain anti-ICOS antibodies can reducethe growth of malignant T cells and/or to determine suitable dosages.

To determine whether an anti-ICOS antibody's ability to reduce tumorgrowth is dependent FcγR, the above described in vivo tumor growth assaywould be performed using SCID mice with compromised Fcγ receptoractivity (e.g., FcRγ^(−/−)). Through a process of interbreeding SCIDmice with mice lacking expression of certain FcγR, SCID mice can begenerated that also lack expression of certain FcγR (e.g., SCID,FcRγ^(−/−) mice). Such mice can be used in assays to assess the abilityof anti-ICOS antibodies to reduce tumor growth through pathways thatinvolve FcγR expression, e.g., ADCC. Based on the results, anti-ICOSantibodies with increased ADCC can be engineered using the techniquesdescribed above. Such antibodies can in turn be used in the compositionsand methods of the invention for the treatment of human T cell disordersand diseases including, but not limited to, autoimmune diseases ordisorders, inflammatory diseases or disorders, and T cell malignancies.

7.8.1. IC9G1-aFuc Binding to Fcgamma Receptors.

The equilibrium binding constants of IC009, IC9G1 and IC9G1-aFuc tohuman and cynomolgus FcγRIIIA-V158, FcγRIIIA-F158, FcγRIIA and FcγRIIBare measured on a BIAcore 3000 instrument (Uppsala, Sweden). Themeasurements are performed according to standard protocols. Briefly, allIgGs are immobilized onto separate flow cells of two CM5 sensor chipsusing standard amino coupling chemistry as recommended by themanufacturer. Immobilized IgG levels range from 8194 to 8725 RUs. Stocksolutions of the recombinantly expressed extracellular domains of allFcγRs at either 4000 or 16000 nM are prepared and then serially diluteddown to the desired concentrations using the instrument buffer (50 mMHBS buffer containing 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA and0.005% P-20). Duplicate injections of each concentration of FcγR aredthen injected over all of the IgG surfaces at a flow rate of 5 μL/min.Binding data are collected for approximately 50 min, followed by a 30sec. pulse of 5 mM HCl between injections to regenerate the IgGsurfaces. Several buffer injections are also interspersed throughout theinjection series. One of these buffer injections is used along with thereference cell data to correct the raw data sets. After all binding datais collected, individual data sets are averaged for each yconcentration, then fit to a 1:1 binding isotherm from which theequilibrium binding constants, K_(D), are derived. Analysis is carriedout using the BIAevaluation software. The K_(D) values (nM) arepresented in FIG. 2.

7.9. IC9G1-aFuc Inhibits Anti-CD3/ICOSL Induced Human T CellProliferation

96-well tissue culture plates are coated with 25 microL 2 μg/ml B7h-Fcprotein and 25 microL of 0.2 μg/ml anti-CD3 antibody (OKT3). Isolated Tcells are plated on the pre-coated plates in the presence of variousconcentration (0.1-20 μg/ml) of IC009, IC9G1 and IC9G1-aFuc antibody. Tcell proliferation is ascertained by measuring after 72 hours ofincubation the number of viable cells in each well using a luminescenceassay. The proliferation of T cells in uncoated wells, and wells coatedwith either anti-CD3 antibody or B7h-Fc protein alone is determined as acontrol.

An example of the results obtained is shown in FIG. 3. Unstimulated Tcells or T cells stimulated by anti-CD3 or B7h-Fc alone displayed a verylimited baseline proliferation. T cell induction by surface boundanti-CD3 and B7h-Fc in the presence of 0.01 mg/ml antibody resulted incell proliferation significantly above the baseline. Anti-CD3/B7h-Fcinduced T cell proliferation is inhibited by all three anti-ICOSantibody tested (IC009, IC9G1 and IC9G1-aFuc) in a dose dependentmanner. The inhibitory activity of IC009, IC9G1 and IC9G1-aFucantibodies was substantially identical in the assay.

7.10. IC9G1-aFuc Does not Inhibit anti-CD3/anti-CD28 Induced Human TCell Proliferation

96-well tissue culture plates are coated with anti-CD3 (OKT3) andanti-CD28 antibodies. Isolated tonsillar T cells are plated on thepre-coated plates in the presence of 10 μg/ml of IC9G1-aFuc antibody. Tcell proliferation is ascertained by measuring after 72 hours ofincubation the number of viable cells in each well using a luminescenceassay. The proliferation of T cells in uncoated wells, and wells coatedwith either anti-CD3 or anti-CD28 antibody only is determined as acontrol.

An example of the results obtained is shown in FIG. 4. Unstimulated Tcells or T cells stimulated by anti-CD3 or anti-CD28 antibody alonedisplayed a very limited baseline proliferation. T cell induction bysurface bound anti-CD3 and anti-CD28 antibodies resulted in cellproliferation significantly above the baseline (aCd3+aCD28). TheIC9G1-aFuc antibody (10 μg/ml) did not inhibit the anti-CD3/anti-CD28induced T cell proliferation (αCd3+αCD28+IC9G1-aFuc).

7.11. IC9G1-aFuc has Enhanced ADCC Activity

The ADCC activity of IC9G1-aFuc is ascertained by an in vitro ADCC assayusing various ICOS expressing primary cells and cell lines. The ADCCassays are performed following a standard protocol. Briefly, targetcells and effector cells (e.g., transgenic NK cells expressing CD16 andassociated signaling polypeptide FCεRI-γ) are plated at a predeterminedratio (e.g., 2.5:1 effector to target ratio) in the presence of theIC9G1-aFuc antibody. The plates are incubated for a pre-determinedlength of time (e.g. 4 hrs). Cell death is ascertained by measuring LDHrelease into the supernatant using a commercially available LDHdetection kit. Antibody mediated cytotoxicity is calculated bysubtracting from the LDH levels detected in the antibody containingwells the background LDH levels detected in antibody-free control wells.Antibody mediated cytotoxicity is expressed as a % of maximumcytotoxicity achievable. The maximum cytotoxicity value is derived fromthe LDH levels measured in wells containing chemically lysed cells (e.g.Triton-X 100 treated well). ADCC activity is presented by plottingantibody mediated cytotoxicity as a function of the antibodyconcentration. EC50 values correspond to the antibody concentrationresulting in a 50% maximum antibody mediated cytotoxicity in theparticular assay.

FIG. 5A shows an example of ADCC activity measurements performed usingstable transfectant HPB-ALL hICOS cells as target cells. The generationof human ICOS transgenic HPB-ALL cell line is described in U.S. Pat. No.6,803,039. The ADCC assay was performed using CD 16/FCεRI-γ transgenicNK cells as effector cells at a 2.5:1 effector to target ratio. The ADCCreaction was allowed to proceed for 4 hrs. The ADCC activity of IC009,IC9G1 and IC9G1-aFuc antibodies was ascertained. IC009 mediated ADCCactivity was below the detection level. The ADCC activity of IC9G1-aFucwas significantly higher than that of the IC9G1 antibody. The EC50values of IC9G1-aFuc and IC9G1 antibodies were 138 pM and 648 pM,respectively, in this assay.

FIG. 5B shows an example of ADCC activity measurements performed usinghuman ICOS transgenic Jurkat cells as target cells. The ADCC assay wasperformed using CD16/FCεRI-γ transgenic NK cells as effector cells at a2.5:1 effector to target ratio. The ADCC reaction was allowed to proceedfor 4 hrs. The ADCC activity of IC009, IC9G1 and IC9G1-aFuc antibodieswas ascertained. All three antibodies displayed measurable ADCCactivity. Maximum % ADCC activity of IC9G1-aFuc and IC9G1 was higherthen that of IC009. Maximum % ADCC activity of IC9G1-aFuc and IC9G1 weresubstantially identical. The EC50 values of IC9G1-aFuc and IC9G1antibodies were 5.7 pM and 61 pM, respectively, in this assay.

FIG. 7 shows an example of ADCC activity measurements performed usingisolated human tonsillar T cells as target cells. ICOS expression ofhuman tonsillar T cells was restricted to the CD4+CD45RO+CD45RA—CXCR5+memory T_(FH) cell population (FIG. 6). Human tonsillar T cells wereisolated with the help of a commercially available kit (Miltenyi MACShuman PanT cell isolation kit). The ADCC assay was performed usingisolated human NK cells as effector cells ar a 2:1 effector to targetratio; the reaction was incubated overnight. The ADCC activity of IC009,IC9G1 and IC9G1-aFuc antibodies was ascertained. IC009 mediated ADCCactivity was slightly above detection level. The IC9G1-aFuc and IC9G1antibodies displayed a dose dependent ADCC activity in this assay. TheADCC activity of IC9G1-aFuc was significantly higher than that of theIC9G1 antibody. The EC50 values of IC9G1-aFuc and IC9G1 antibodies were8.2 pM and 60.4 pM, respectively, in this assay.

FIG. 8 shows an example of ADCC activity measurements performed usingisolated cynomolgus splenic T cells as target cells. ICOS expression wassubstantially restricted to the CD4+CD45RA− memory T cell population inthe spleen (FIG. 8A). Cynomolgus splenic T target cells were isolatedusing a non-human primate T cell isolation kit (Miltenyi). The ADCCassay was performed using isolated human NK cells as effector cells ar a2:1 effector to target ratio; the reaction was incubated overnight. TheADCC activity of IC009, IC9G1 and IC9G1-aFuc antibodies was ascertained.IC009 mediated ADCC activity was below detection level. The IC9G1-aFucand IC9G1 antibodies displayed a dose dependent ADCC activity in thisassay. The ADCC activity of IC9G1-aFuc was significantly higher thanthat of the IC9G1 antibody. The EC50 values of IC9G1-aFuc and IC9G1antibodies were 14.6 pM and 236 pM, respectively, in this assay.

FIG. 8 shows an example of ADCC activity measurements performed usingisolated cynomolgus mesenteric lymph node (MLN) T cells as target cells.ICOS expression was substantially restricted to the CD4+CD45RA−activated T cell population in the MLN (FIG. 9A). Cynomolgus splenic Ttarget cells were isolated using a non-human primate T cell isolationkit (Miltenyi). The ADCC assay was performed using isolated human NKcells as effector cells ar a 2:1 effector to target ratio; the reactionwas incubated overnight. The ADCC activity of IC009, IC9G1 andIC9G1-aFuc antibodies was ascertained. IC009 mediated ADCC activity wasat detection level. The IC9G1-aFuc and IC9G1 antibodies displayed a dosedependent ADCC activity in this assay. The ADCC activity of IC9G1-aFucwas significantly higher than that of the IC9G1 antibody. The EC50values of IC9G1-aFuc and IC9G1 antibodies were 17.1 pM and 198 pM,respectively, in this assay.

7.12. Pharmacokinetic Profile of IC9G1-aFuc in Cynomolgus Monkeys

Cynomolgus monkeys were administered a single IV dose of IC9G1-aFucantibody on day 0 of the experiment. Experimental design is outlined inTable 3.

TABLE 3 Experimental design of in vivo studies of IG9G1-aFuc incynomolgus monkeys. Group Agent Dose (mg/kg) Number 1 Carrier only 0 5males 2 IC9G1-aFuc 0.01 5 males 3 IC9G1-aFuc 0.1 5 males 4 IC9G1-aFuc 15 males 5 IC9G1-aFuc 10 5 males 6 IC009 10 5 males

The pharmacokinetic profile of IC9G1-aFuc was analyzed by delivering asingle dose of the antibody and monitoring its serum concentration overtime. Serum concentration of IC9G1-aFuc was measured by ELISA accordingto standard protocols. ICG91-aFuc serum concentration as a function oftime is presented in FIG. 10. Systemic exposure based on estimates ofAUC_(LAST) for IC9G1-aFuc and Cmax increased in a dose proportionalmanner with increasing the dose, reflecting the linearity in theantibody pharmacokinetic properties. Mean terminal half-life (t½ lz)values of 4.36±1.52 days, 6.34±1.44 days and 7.87±1.09 days wereobserved following bolus infusions of 0.1 mg/kg, 1 mg/kg and 10 mg/kg,respectively.

7.13. In vivo T Cell Depletion following the Administration of a SingleDose of IC9G1-aFuc

Cynomolgus monkeys were administered a single IV dose of IC9G1-aFucantibody. Antibody dose administered to the various animals is describedin Table 3. Two animals from each group were sacrificed on day 8post-dosing. Three animals from each group were sacrificed on day 29post-dosing. The level of circulating, splenic and mesnteric lymph node(MLN) ICOS+ T cells were monitored for four weeks following the deliveryof the single antibody dose. ICOS+ T cells were monitored by flowcytometry.

FIG. 11 shows the changes in circulating ICOS+ memory T cell levelfollowing the administration of a single dose of IC9G1-aFuc antibody.Circulating memory T cells were defined as CD3+CD4+CD45RA-ICOS+ cellsfor the purposes of this study. Absolute numbers of circulating memory Tcells detected were normalized to the circulating memory T cell numbersdetected on day 0 prior to antibody administration. Administration of asingle dose of 0.01 mg/kg of IC9G1-aFuc antibody resulted in asignificant reduction in circulating memory T cell count by day 4.Administration of a single dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg ofIC9G1-aFuc antibody resulted in the complete elimination of circulatingmemory T cells by day 4 of the experiment. Recovery of the circulatingmemory T cell compartment over time was dose dependent.

FIG. 13 provides an example of the depletion results seen in themesenteric lymph node (MLN) T cell compartment. MLN T cells wereisolated from animals sacrificed on day 8 and day 29 of the experiment.Absolute numbers of ICOS+ memory helper T cells isolated from the MLNwere determined by flow cytometry. Memory helper T cells were defined asCD3+CD4+CD45RA− for the purposes of the experiment. Absolute numbers ofICOS+ memory T cells isolated from the MLN on day 8 are displayed inFIG. 13A. Administration of a single dose of 0.1 mg/kg and 10 mg/kg ofIC9G1-aFuc antibody resulted in a significant dose dependent depletionof ICOS+ memory helper T cells from the mesenteric lymph node. Similardepletion of ICOS+ memory helper T cells was detected in the tonsil andmandibular lymph node. FIG. 13B presents the % depletion of ICOS+ memoryhelper T cells in the mesenteric lymph node on day 8. % depletion wascalculated by normalizing the absolute ICOS+ memory helper T cellnumbers detected in IC9G1-aFuc treated animals to the cell numbersdetected in the carrier only treated control animals. Administration ofa single dose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFuc antibody resultedin the depletion of greater than 60% and 90%, respectively, of ICOS+memory helper T cells from the mesenteric lymph node by day 8.

FIG. 14 provides an example of the depletion results seen in the splenicT cell compartment. Splenic T cells were isolated from animalssacrificed on day 8 and day 29 of the experiment. Absolute numbers ofsplenic ICOS+ memory helper T cells were determined by flow cytometry.Memory helper T cells were defined as CD3+CD4+CD45RA− for the purposesof the experiment. Absolute numbers of ICOS+ memory T cells isolatedfrom the MLN on day 8 and 29 are displayed in FIG. 14A. Administrationof a single dose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFuc antibodyresulted in a significant depletion of splenic ICOS+ memory helper Tcells by day 8. The recovery of splenic ICOS+ memory helper T cells weredose dependent; splenic ICOS+ T cell recovery on day 28 was morepronounced in animals dosed with 0.1 mg/kg IC9G1-aFuc than in animalsdosed with 10 mg/kg IC9G1-aFuc. FIG. 14B presents the % depletion ofsplenic ICOS+ memory helper T cells in the mesenteric lymph node on day8 and 29. % depletion was calculated by normalizing the absolute ICOS+memory helper T cell numbers detected in IC9G1-aFuc treated animals tothe cell numbers detected in the carrier only treated control animals.Administration of a single dose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFucantibody resulted in the depletion of greater than 60% of splenic ICOS+memory helper T cells by day 8. By day 29, the splenic ICOS+memoryhelper T cell compartment of animals dosed with 0.1 mg/kg of IC9G1-aFucstarted to recover. The splenic ICOS+ memory helper T cell compartmentof animals dosed with 10 mg/kg of IC9G1-aFuc was more depleted on day 29than on day 8.

7.14. In Vivo Administration of a Single Dose of IC9G1-aFuc Results inthe Dissolution of Already Formed Germinal Centers

Cynomolgus monkeys were administered a single IV dose of IC9G1-aFucantibody. Antibody dose administered to the various animals is describedin Table 3. Two animals from each group were sacrificed on day 8post-dosing. Three animals from each group were sacrificed on day 29post-dosing. The architecture of splenic white pulp was examined on day8 and 29 using standard histology protocols. The number of mesentericlymp node and splenic germinal center B cells were measured on day 8 and29 by flow cytometry.

FIG. 15 presents an example of the architectural changes to the splenicwhite pulp caused by the administration of a single IV dose ofIC9G1-aFuc. Low (10×) and high (20×) magnification of histologicalsections of white pulp isolated from control and IC9G1-aFuc dosedanimals on day 8 (FIG. 15A) and day 29 (FIG. 15B) of the experiment isshown. Splenic follicles were atrophied on day 29 after administrationof a single dose of IC9G1-aFuc antibody to cynomolgus monkeys. Themorphology of splenic white pulp was examined following theadministration of a single dose f IC9G1-aFuc antibody. Histologicalsections of the spleen isolated on day 8 (A) and day 29 (B) afterIC9G1-aFuc antibody administration are shown. IC9G1-aFuc administrationresults in severe atrophy of splenic follicles by day 29.

FIG. 12 shows the flow cytometry protocol that was used to identifygerminal center B cells. Lymphocytes were isolated from lymphatic organsof sacrificed animals following standard protocols. Isolated lymphocyteswere immunostained with anti-CD3, anti-CD20, anti-IgM and anti CD95 oranti-CD27 antibodies. Dead cells were excluded from the analysis withthe aid of 7AAD staining. Germinal center N cells were defined as eitherCD3-CD20+IgM−CD95+ or CD3-CD20+IgM−CD27+ cells.

FIG. 13 provides an example of the effects on the mesenteric lymph node(MLN) germinal center B cell compartment caused by the administration ofa single dose of IC9G1-aFuc. MLN lymphocytes were isolated from animalssacrificed on day 8 and day 29 of the experiment. Absolute numbers ofgerminal center B cells were determined by flow cytometry. Germinalcenter B cells were defined as CD20+IgM−CD95+ cells for the purposes ofthe experiment. Absolute numbers of germinal center B cells isolatedfrom the MLN on day 8 are displayed in FIG. 13A. Administration of asingle dose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFuc antibody resulted ina significant dose dependent loss of germinal center B cells from themesenteric lymph node. The administration of a single dose of the IC009antibody resulted in a comparable loss of germinal center B cells fromthe MLN on day 8. FIG. 13B presents the % dissolution of germinalcenters in the mesenteric lymph node on day 8. % dissolution wascalculated by normalizing the absolute germinal center B cell numbersdetected in IC9G1-aFuc treated animals to the cell numbers detected inthe carrier only treated control animals. Administration of a singledose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFuc antibody resulted in thedissolution of greater than 75% and 90%, respectively, of germinalcenters from the mesenteric lymph node by day 8. Administration of asingle dose of 10 mg/kg of IC009 antibody resulted in the dissolution ofgreater than 80% of germinal centers from the mesenteric lymph node byday 8. The germinal center B cells in this model system were present inthe MLN prior to administration of the IC9G1-aFuc antibody. The loss ofgerminal center B cells from the MLN therefore indicates that thedepletion of ICOS+ memory helper T cells leads to the dissolution ofpreviously formed germinal centers.

FIG. 14 provides an example of the effects on the splenic germinalcenter B cell compartment caused by the administration of a single doseof IC9G1-aFuc. Splenic lymphocytes were isolated from animals sacrificedon day 8 and day 29 of the experiment. Absolute numbers of germinalcenter B cells were determined by flow cytometry. Germinal center Bcells were defined as CD20+IgM−CD95+ cells for the purposes of thisexperiment. Absolute numbers of germinal center B cells isolated fromthe spleen on day 8 and 29 are displayed in FIG. 14A. Administration ofa single dose of 0.1 mg/kg and 10 mg/kg of IC9G1-aFuc antibody did notsignificantly affect splenic germinal center B cells numbers on day 8.By day 29, however, the splenic germinal center B cell numbers weresignificantly reduced in IC9G1-aFuc treated animals, In contrast, nosignificant change in splenic germinal center B cell numbers weredetected in IC009 treated animals. FIG. 14B presents the % dissolutionof splenic germinal centers on day 8 and 29. % dissolution wascalculated by normalizing the absolute germinal center B cell numbersdetected in IC9G1-aFuc treated animals to the cell numbers detected inthe carrier only treated control animals. Administration of a singledose of IC9G1-aFuc antibody did not result in significant dissolution ofsplenic germinal centers by day 8. By day 29, however, approximately 80%of splenic germinal centers were dissolved in the IC9G1-aFuc treatedanimals. No significant dissolution of splenic germinal centers weredetected on either day 8 or 29 following the administration of 10 mg/kgof IC009. The germinal center B cells in this model system were presentin the spleen prior to administration of the IC9G1-aFuc antibody. Theloss of germinal center B cells from the spleen therefore indicates thatthe depletion of ICOS+ memory helper T cells leads to the dissolution ofpreviously formed germinal centers.

7.15. ICOS and ICOSL mRNA Expression is Elevated in Patients Affected byInflammatory or Autoimmune Diseases 7.16. ICOS is a Therapeutic Targetin Systemic Lupus Erythematosus

Inducible costimulator (ICOS) is involved in the regulation ofautoimmune and proinflammatory responses and may play important roles inthe pathogenesis of SLE. We used a genomics approach to evaluate themRNA expression levels of a panel of cytokines and immune regulators inlesional skin of active SLE patients with cutaneous involvement.

We profiled lesional skin and whole blood (WB) from a large panel of SLEpatients with cutaneous involvement using the Affymetrix® human wholegenome array (WGA) platform. TaqMan® QRT-PCR using a BioMark™ 48.48dynamic array from Fluidigm was used to measure the mRNA levels of bothlong and short alternative splicing forms of ICOS, along with a largepanel of cytokines.

ICOS mRNA was overexpressed in lesional skin for approximately 50-60% ofthe SLE patients evaluated in the study (FIG. 20). Positive correlationsbetween ICOS and the ICOS ligand mRNA overexpression, and between ICOSand IL-10 mRNA overexpression were observed. Robust overexpression ofthese mRNAs was not observed in peripheral uninvolved tissues of SLEpatients. Additionally, we used TaqMan QRT-PCR to determine whether theshort or long alternative splicing form of ICOS is overexpressed in SLE,and also evaluated miR-101 expression in ICOS+ memory T cells purifiedfrom WB of SLE patients.

Two protein isoforms of ICOS were identified from the cDNA database (seeFIG. 16). Full length of ICOS (SEQ ID NO:32) has 199 amino acids. Itcontains a signal peptide, an extracellular domain, a transmembranedomain and a cytoplasmic domain. In the cytoplasmic domain, it containsYMFM (residues 180-183 of SEQ ID NO:32) conserved motif for PI3Kbinding. The short form of ICOS (SEQ ID NO:33) has 168 amino acids. Theshort form has a in frame truncation in cytoplasmic domain caused byexon 4 skipping. The truncation generated a much shorter cytoplasmicdomain and lost PI3K binding site that may have functional impacts onICOS function.

In silico analysis of ICOS 3′ UTR (residues 238-2284 of SEQ ID NO:34)using a MiRanda revealed several putative miRNA target sites (FIG. 17).MicroRNA target region one (MTR1), a 47 bp region containing targetsequences for miR-101, 103/107 and 338, and miRNA target region two(MTR2), a 47 bp region containing the target sequence for miR-149.Complementarity of ICOS cDNA and the identified miRNA molecules is shownin FIG. 17. (Di Yu, & Carola G. Vinuesa et al. Nature (2007) 450,299-303).

Affymetrix GeneChip and qRT-PCR Profiling—SLE: We profiled lesional skinand whole blood (WB) from a panel of SLE patients with cutaneousinvolvement using the Affymetrix® human whole genome array (WGA)platform. TaqMan® qRT-PCR using a BioMark™ 48.48 dynamic array fromFluidigm was used to measure the mRNA levels of ICOS, along with a largepanel of cytokines.

FIG. 20A shows ICOS and ICOSL mRNA relative expression (log 2 scale) inSLE (Systemic Lupus Erythematosus) skin lesion specimens. Individualfold-change values were determined relative to a normal skin samplecontrol. Data was generated on Fluidigm's BioMark™ 48.48 dynamic array.Bars represent mean of relative expression (fold-change) for eachtranscript (ICOS or ICOSL) examined.

Raw signal intensity values (log 2 scale) for CD4 (FIG. 20B) and CD3εmRNA (FIG. 20C) in normal and SLE (Systemic Lupus Erythematosus) skinspecimens. Data (GC-RMA normalized) was generated on the AffymetrixHuman Genome U133 Plus 2.0 Array. Bars represent mean of raw signalintensity for normal and SLE samples.

FIG. 21A: CD28, CTLA4, ICOS and ICOSL mRNA relative expression (log 2scale) in SLE (Systemic Lupus Erythematosus) whole blood specimens.Individual fold-change values were determined relative to a poolednormal whole blood sample control. Data was generated on Fluidigm'sBioMark™ 48.48 dynamic array. Bars represent mean of relative expression(fold-change) for each transcript (CD28, CTLA4, ICOS or ICOSL) analyzed.

Raw signal intensity values (log 2 scale) for CD4 (FIG. 21B) and CD3εmRNA (FIG. 21C).in normal and SLE (Systemic Lupus Erythematosus) wholeblood specimens. Data (GC-RMA normalized) was generated on theAffymetrix Human Genome U133 Plus 2.0 Array. Bars represent mean of rawsignal intensity for normal and SLE samples.

7.17. ICOS Expression in Inclusion Body Myositis (IBM) andDermatomyositis (DM)

Inducible costimulator (ICOS), a receptor on activated T-cells, plays acentral role in humoral immunity. Elevated levels of ICOS are present inpatients with autoimmune diseases (e.g., rheumatoid arthritis andsystemic lupus) and effector cytokines have been shown to correlate withincreased levels of this protein. We used genomics technologies toinvestigate the over-expression of ICOS and the ICOS ligand (ICOSL) inmuscle tissue taken from patients with inclusion body myositis (IBM),dermatomyositis (DM) and polymyositis (PM) and present resultsconsistent with a regulatory mechanism of ICOS by the T-cell expressedmiRNA, miR-101.

We profiled muscle specimens from myositis patients using TaqMan®QRT-PCR (Fluidigm's BioMark™ 48.48 dynamic array). mRNAs (noncoding RNAsexpressed by T lymphocytes and known to regulate gene expression) thatpotentially regulate ICOS were identified by 2 criteria: (1) theirsequences were complementary to the 3′ UTR region of ICOS, and (2) theywere significantly differentially expressed in the opposite direction ofICOS mRNA in IBM, PM and DM muscle, compared with normal control muscle.

ICOS mRNA in IBM muscle specimens were highly up-regulated by an averageof 40-fold, with mRNAs of ICOSL up-regulated by an average of 3.5-fold,compared to normal controls. In DM muscle specimens, ICOS mRNAs wereup-regulated by an average of 5-fold; ICOSL mRNA showed no significantupregulation compared with normal controls. ICOS mRNA in IBM musclespecimens were highly up-regulated (over 70 fold upregulation), withICOSL mRNAs up-regulated by 2-fold, compared to normal controls.Overexpression of ICOS and ICOSL mRNA was not observed in whole bloodfrom IBM or DM muscle. CD4 and CD3ε mRNAs were strongly over-expressedin IBM muscle specimens, whereas only CD4 mRNA was over-expressed inmuscle specimens of DM patients. The presence of ARE sites (AU richregion for protein binding) and sequence complementarity between the 3′UTR domain in ICOS and miR-101 suggest that miR-101 is a potentialregulator of ICOS. We subsequently evaluated the expression level ofmiR-101, as well as the feasibility of this miRNA to regulate thistranscript. The expression of miR-101 was significantly down-regulatedby an average of 4-fold and 2.5-fold, respectively, in IBM and DM musclecompared with normal control muscle.

ICOS mRNA is overexpressed in muscle tissue from IBM, DM and PMpatients. Strong over-expression of mRNAs of CD4 and CD3ε suggest anincrease in CD4+ T cell infiltration at the disease site of IBM patientsas has been previously noted. The significant under-expression ofmiR-101 in muscle tissue from IBM and DM patients confirmed theobservation from sanroque mice previously reported.

Affymetrix GeneChip, qRT-PCR and microRNA Profiling—Myositis: Weprofiled muscle biopsy and whole blood (WB) from a panel of myositispatients using the Affymetrix® human whole genome array (WGA) platform.Additionally, we profiled muscle specimens from myositis patients usingboth TaqMan® qRT-PCR (Fluidigm's BioMark™ 48.48 dynamic array) and theApplied Biosystem MicroRNA TaqMan Human MicroRNA Array v1.0 platforms.mRNAs (noncoding RNAs expressed by T lymphocytes and known to regulategene expression) that potentially regulate ICOS were identified by 2criteria: (1) their sequences were complementary to the 3′ UTR region ofICOS, and (2) they were significantly differentially expressed in theopposite direction of ICOS mRNA in IBM, PM and DM muscle, compared withnormal control muscle.

FIG. 18 shows miR-101 relative expression in muscle specimens frommyositis patients (IBM=Inclusion-body myositis, PM=polymyositis,DM=Dermatomyositis). Individual expression values were determinedrelative to a normal muscle sample control. Data was generated on ABI'sHuman MicroRNA Array v1.0 platform. Bars represent mean of relativeexpression for each disease sub-type.

FIG. 19A shows ICOS and ICOSL mRNA relative expression (log 2 scale) inmyositis muscle specimens (IBM=Inclusion-body myositis, PM=polymyositis,DM=Dermatomyositis). Individual fold-change values were determinedrelative to a normal muscle sample control. Data was generated onFluidigm's BioMark™ 48.48 dynamic array. Bars represent mean of relativeexpression (fold-change) for each disease sub-type and transcript (ICOSor ICOSL) combination.

Raw signal intensity values (log 2 scale) for CD4 (FIG. 19B) and CD3ε(FIG. 19C) mRNA in normal and myositis muscle specimens(IBM=Inclusion-body myositis, PM=polymyositis, DM=Dermatomyositis). Data(GC-RMA normalized) was generated on the Affymetrix Human Genome U133Plus 2.0 Array. Bars represent mean of raw signal intensity for normalsand each disease sub-type.

FIG. 19D shows ICOS and ICOSL mRNA relative expression (log 2 scale) inmyositis whole blood samples (IBM=Inclusion-body myositis,PM=polymyositis, DM=Dermatomyositis). Individual fold-change values weredetermined relative to a normal muscle sample control. Data wasgenerated on Fluidigm's BioMark™ 48.48 dynamic array. Bars representmean of relative expression (fold-change) for each disease sub-type andtranscript (ICOS or ICOSL) combination.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated anti-ICOS antibody comprising a VH domain, a VK domainand a variant Fc region, wherein the antibody mediates enhanced ADCCactivity as compared to the level of ADCC activity mediated by a parentantibody comprising the VH and VK domains and a wild type Fc region,wherein said antibody is capable of depleting circulatingICOS-expressing T cells in a mammal for at least 14 days whenadministered as a single dose of 125 mg/m², and wherein the variant Fcregion is either: a. a variant Fc region comprising at least onesubstitution of an amino acid residue selected from the group consistingof: residue 239, 330, and 332, wherein the amino acid residue positionsare determined according to the EU convention; or b. an engineered Fcregion, wherein the engineered Fc region comprises complexN-glycoside-linked sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain.
 2. Theantibody of claim 1, wherein the EC50 of the antibody as measured in anin vitro ADCC assay is at least about 7×lower than the EC50 value of theparent antibody.
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)7. (canceled)
 8. (canceled)
 9. (canceled)
 10. The antibody of 1, whereinthe VH domain comprises the amino acid sequence of SEQ ID NO:7 and theVK domain comprises the amino acid sequence of SEQ ID NO:2.
 11. Anucleic acid encoding the amino acid sequence of the antibody of claim 1wherein said nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, andSEQ ID NO:31.
 12. (canceled)
 13. A vector comprising the nucleic acid ofclaim
 11. 14. (canceled)
 15. An isolated cell comprising the vector ofclaim
 13. 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)20. (canceled)
 21. A pharmaceutical composition comprising the antibodyof claim 1 in a pharmaceutically-acceptable carrier.
 22. (canceled) 23.A method of treating an autoimmune disease or disorder or aninflammatory disease or disorder in a human, comprising administering toa human in need thereof a therapeutically-effective amount of theantibody of claim
 1. 24. The method of claim 23, wherein the autoimmunedisease or disorder is SLE or scleroderma.
 25. (canceled)
 26. (canceled)27. (canceled)
 28. (canceled)
 29. The method of claim 23, wherein theinflammatory disease or disorder is inclusion-body myositis (IBM),polymyositis (PM) or dermatomyositis (DM).
 30. A method of depletingICOS expressing T cells in a human patient comprising administering to ahuman in need thereof a therapeutically-effective amount of the antibodyof claim
 1. 31. The method of claim 30, wherein the depletionsubstantially persists for at least about 1, at least about 2, at leastabout 3 or at least about 4 weeks following the administration of theantibody.
 32. The method of claim 30, wherein at least about 95% of theT cells are depleted.
 33. The method of claim 30, wherein the ICOSexpressing T cell is a memory T cell.
 34. The method of claim 30,wherein the ICOS expressing T cell is a circulating T cell. 35.(canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled)
 39. (canceled)40. (canceled)
 41. A method of depleting circulating class switched Bcells in a human patient comprising administering an effective amount ofthe antibody of claim
 1. 42. (canceled)
 43. (canceled)
 44. The method ofclaim 41, wherein the depletion substantially persists for at leastabout 1, at least about 2, at least about 3 or at least about 4 weeksfollowing the administration of the antibody.
 45. The method of claim41, wherein at least about 95% of the circulating class switched B cellsare depleted.
 46. (canceled)
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. (canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled)54. (canceled)
 55. (canceled)
 56. (canceled)
 57. (canceled) 58.(canceled)
 59. An isolated anti-ICOS antibody comprising a VH domain, aVK domain and a modified Fc region, wherein the antibody has complexN-glycoside-linked sugar chains bound to the modified Fc region in whichfucose is not bound to N-acetylglucosamine in the reducing end in thesugar chain, wherein the antibody mediates enhanced ADCC activity ascompared to the level of ADCC activity mediated by a parent antibodycomprising the VH and VK domains and a non-engineered Fc region, andwherein said antibody is capable of depleting class switched B cells ina primate, and wherein the modified Fc region is either a. a variant Fcregion comprising at least one substitution of an amino acid residueselected from the group consisting of: residue 239, 330, and 332,wherein the amino acid residue positions are determined according to theEU convention; or b. an engineered Fc region, wherein the engineered Fcregion comprises complex N-glycoside-linked sugar chains in which fucoseis not bound to N-acetylglucosamine in the reducing end in the sugarchain.
 60. The antibody of claim 59, wherein the primate is a non-humanprimate.
 61. The antibody of claim 59, wherein the primate is a human.62. The antibody of claim 59, wherein the depletion substantiallypersists for at least about 1, at least about 2, at least about 3 or atleast about 4 weeks following the administration of the antibody. 63.The antibody of claim 59, wherein at least about 95% of the circulatingclass switched B cells are depleted.